Co-reporter:Yanying Li, Xiaodan Liu, Xiaoyan Dong, Lin Zhang, and Yan Sun
Langmuir July 22, 2014 Volume 30(Issue 28) pp:8500-8508
Publication Date(Web):June 29, 2014
DOI:10.1021/la5017438
Virus-like particle (VLP) of murine polyomavirus (MPV) is a T = 7d icosahedral capsid that self-assembles from 72 capsomeres (Caps), each of which is a pentamer of major coat protein VP1. VLP has great potential in vaccinology, gene therapy, drug delivery, and materials science. However, its application is hindered by high cost downstream processes, leading to an urgent demand of a highly efficient affinity ligand for the separation and purification of Cap by affinity chromatography. Herein a biomimetic design strategy of an affinity peptide ligand of Cap has been developed on the basis of the binding structure of the C-terminus of minor coat protein (VP2-C) on the inner surface of Cap. The molecular interactions between VP2-C and Cap were first examined using all-atom molecular dynamics (MD) simulations coupled with the molecular mechanics/Poisson–Boltzmann surface area (MM/PBSA) method, where V283, P285, D286, W287, L289, and Y296 of VP2-C were identified as the hot spots. An affinity peptide library (DWXLXLXY, X denotes arbitrary amino acids except cysteine) was then constructed for virtual screening sequently by docking with AUTODOCK VINA, binding structure comparison, and final docking with ROSETTA FlexPepDock. Ten peptide candidates were selected and further confirmed by MD simulations and MM/PBSA, where DWDLRLLY was found to have the highest affinity to Cap. In DWDLRLLY, six residues are favorable for the binding, including W2, L4, L6 and Y8 inheriting from VP2-C, and R5 and L7 selected in the virtual screening. This confirms the high efficiency and accuracy of the biomimetic design strategy. DWDLRLLY was then experimentally validated by a one-step purification of Cap from crude cell lysate using affinity chromatography with the octapeptide immobilized on Sepharose gel. The purified Caps were observed to self-assemble into VLP with consistent structure of authentic MPV.
Co-reporter:Baolong Xie, Xi Li, Xiao-Yan Dong, and Yan Sun
Langmuir August 19, 2014 Volume 30(Issue 32) pp:9789-9796
Publication Date(Web):August 1, 2014
DOI:10.1021/la5025197
Alzheimer’s disease (AD) is the most prevalent form of dementia, and aggregation of amyloid β-proteins (Aβ) into soluble oligomers and fibrils has been implicated in the pathogenesis of AD. Herein we developed acidulated serum albumin for the inhibition of Aβ42 fibrillogenesis. Bovine serum albumin (BSA) was modified with diglycolic anhydride, leading to the coupling of 14.5 more negative charges (carboxyl groups) on average on each protein surface. The acidulated BSA (A-BSA) was characterized and confirmed to keep the tertiary structure and stability of BSA. Extensive biophysical and biological analyses showed that A-BSA significantly inhibited Aβ42 fibrillogenesis and mitigated amyloid cytotoxicity. As compared to the Aβ42-treated group (cell viability, 50%), the cell viability increased to 88% by the addition of equimolar A-BSA. The inhibitory effect was remarkably higher than that of BSA at the same concentration. On the basis of the experimental findings, a mechanistic model was proposed. The model considers that Aβ42 is bound to the A-BSA surface by hydrophobic interactions, but the widely distributed negative charges on the A-BSA surface give rise to electrostatic repulsions to the bound Aβ42 that is also negatively charged. The two well-balanced opposite forces make Aβ42 adopt extended conformations instead of the β-sheet structure that is necessary for the on-pathway fibrillogenesis, even when the protein is released off the surface. Thus, A-BSA greatly slows down the fibrillation and changes the fibrillogenesis pathway, leading to the formation of less toxic aggregates. The findings and the mechanistic model offer new insights into the development of more potent inhibitors of Aβ fibrillogenesis and cytotoxicity.
Co-reporter:Baolong Xie, Fufeng Liu, Xiaoyan Dong, Yongjian Wang, Xiaoguang Margaret Liu, Yan Sun
Journal of Inorganic Biochemistry 2017 Volume 171(Volume 171) pp:
Publication Date(Web):1 June 2017
DOI:10.1016/j.jinorgbio.2017.03.009
•Amyloid β-proteins (Aβ) aggregation is the key process leading to Alzheimer’s disease.•Acidulated serum albumin (A-HSA) was prepared for inhibiting Cu2 +-Aβ42 aggregation.•A-HSA remarkably reduced the cytotoxicity of Cu2 +-Aβ42 aggregates.•A-HSA changed the coordination mode of Cu2 + and Aβ42 from component I to II.•A mechanistic model was developed based on the findings.Aggregation of amyloid β-proteins (Aβ) induced by Cu2 + is a crucial element in the pathogenesis of Alzheimer's disease (AD), and cerebral acidosis is a common complication of AD. Under mildly acidic conditions, Cu2 +-Aβ species have higher tendency to generate neurotoxic aggregates. Hence it is of significance to develop potent agents that inhibit Cu2 +-mediated Aβ aggregation under a mildly acidic condition. Herein we synthesized acidulated human serum albumin (A-HSA) to mitigate Cu2 +-mediated Aβ42 aggregation and cytotoxicity at pH 6.6. Extensive experiments showed that A-HSA altered the pathway of Cu2 +-mediated Aβ42 aggregation and protected SH-SY5Y cells from cytotoxicity and oxidative damage induced by Cu2 +-Aβ42 species. Equimolar A-HSA increased cell viability from 52% to 91% as compared to Cu2 +-Aβ42-treated group. Stopped-flow fluorescence analysis revealed that A-HSA changed the Cu2 +-Aβ42 coordination mode from component I to II on the second timescale at pH 6.6, which avoided the formation of aggregation-prone Cu2 +-Aβ42 aggregates. The findings revealed that the more negative charges on A-HSA surface could stabilize the protonated form of the adjacent histidine residues of Aβ42. Hence, component I, which is necessary to form toxic aggregates, became unstable in the presence of A-HSA. On the other hand, hydrophobic binding and electrostatic repulsion could work simultaneously on the bound Aβ42 on A-HSA surface. The two opposite forces stretched Aβ42 conformations, which inhibited the formation of toxic Cu2 +-Aβ42 aggregates. Thus, A-HSA worked as a bifunctional inhibitor against Cu2 +-mediated Aβ42 aggregation and cytotoxicity under a mildly acidic condition.The research revealed that acidulated human serum albumin (A-HSA) changed the coordination mode of Cu2+ and amyloid β-proteins (Aβ) at pH 6.6, which avoided the formation of aggregation-prone Cu2+-Aβ42 aggregates. Thus, A-HSA worked as a bifunctional inhibitor against Cu2+-Aβ42 aggregation and cytotoxicity under a mildly acidic condition.Download high-res image (87KB)Download full-size image
Co-reporter:Ming Li, Yanying Li, Linling Yu, Yan Sun
Journal of Chromatography A 2017 Volume 1486(Volume 1486) pp:
Publication Date(Web):24 February 2017
DOI:10.1016/j.chroma.2016.11.012
•Poly(allylamine)-grafted Sepharose gels were characterized for protein chromatography.•Both capacity and uptake rate started to increase at ionic capacity of 373 mmol/L.•Drastic increases of capacity and uptake rate were obtained at ionic capacity of 618 mmol/L.•Protein adsorption on the resins exhibited salt tolerance till 500 mmol/L NaCl.•A mild condition of pH 5.0 offered efficient elution of bound proteins.This work reports poly(allylamine) (PAA), as a polymeric ion-exchange ligand for protein chromatography. Sepharose FF was modified with PAA, and six anion exchangers with ionic capacities (ICs) from 165 to 618 mmol/L were prepared. Inverse size exclusion chromatography, adsorption equilibrium, uptake kinetics and column elution were performed. It was found that both the adsorption capacity and effective diffusivity maintained low values in the IC range of 165–373 mmol/L, but they started to increase beyond 373 mmol/L, and increased by 80% and 23 times, respectively, when the IC reached 618 mmol/L. Interestingly, a drastic decrease of pore size was observed around the IC of 373 mmol/L. The results suggest that the PAA chains played an important role in protein adsorption by altering the inner pore structure of the gels. It is considered that, PAA chains turn from inextensible states with multipoint-grafting on the pore surface at low coupling densities (IC<373 mmol/L) to closer, extended and flexible grafting states with less coupling points at higher coupling densities (IC>373 mmol/L). These characters of the grafted chains at higher IC values benefit in protein adsorption by three-dimensional binding and encouraged the happening of “chain delivery” of bound proteins on the chains. Besides, the ion exchangers showed favorable adsorption and uptake properties in a wide ionic strength range, 0–500 mmol/L NaCl, indicating much better salt tolerance feature than the so-far reported ion exchangers. Moreover, a mild condition of pH 5.0 offered effective recovery of bound proteins in elution chromatography. The results indicate that the PAA-based anion exchanger of a high IC value is promising for high-capacity protein chromatography dealing with feedstock of a wide range of ionic strengths.
Co-reporter:Yangyang Zhao, Xiaoyan Dong, Linling Yu, Yan Sun
Journal of Chromatography A 2016 Volume 1427() pp:102-110
Publication Date(Web):4 January 2016
DOI:10.1016/j.chroma.2015.11.084
•Resins with reduced charge densities on poly(ethylenimine) chains were fabricated.•BSA adsorption capacity kept unchanged with decreasing charge density.•BSA uptake rate increased drastically when charge density was lowered to 440 mmol/L.•Salt-sensitivity of protein adsorption increased with decreasing charge density.The adsorption and elution behaviors of bovine serum albumin (BSA) on poly(ethylenimine) (PEI)-grafted Sepharose FF resins were recently studied and a critical ionic capacity (cIC; 600 mmol/L) was found, above which the uptake rate increased drastically due to the occurrence of significant “chain delivery” effect. Moreover, above the cIC value, higher salt concentrations were required for protein elution due to the high charge density of the resins. In this work, we have reduced the charge density on the PEI chains of a PEI-grafted resin by neutralization of the amine groups with sodium acetate. PEI-modified resin with IC of 740 mmol/L (FF-PEI-L740, IC > cIC) was chosen as the starting material, and three resins with residual IC values of 660, 560 and 440 mmol/L (FF-PEI-R440) were obtained. The adsorption and chromatographic behaviors of these resins for BSA were investigated. It was found that, with IC decreasing from 740 to 440 mmol/L, the adsorption capacity kept almost unchanged; the effective protein diffusivity (De) also showed negligible variations as IC decreased from 740 to 560 mmol/L (De/D0 = 0.38 ± 0.04). However, it was interesting to observe a three-fold increase of the De value for FF-PEI-R440 (De/D0 = 1.23 ± 0.08). It is considered that the occurrence of the drastic uptake rate increase in FF-PEI-R440 was attributed to the decreased available binding sites for protein molecule, which led to the decrease of binding strength, thus facilitated the happenings of “chain delivery” effect of bound proteins. Besides, a study on the effect of ionic strength clarified that the lower the IC value, the higher the sensitivity of protein binding to salt concentration due to the easily screened electrostatic interactions at low surface charge densities. The ionic strength at the elution peak also decreased with decreasing IC in accordance with the salt sensitivity order. Column breakthrough studies demonstrated that the dynamic adsorption capacity of FF-PEI-R440 was much higher than the other three resins at flow rates higher than 30 cm/h because of its high uptake rate. The findings in this work provided new insights into the effects of the interactions between proteins and grafted polymers on adsorption equilibria and uptake kinetics, which would help the selection and design of suitable media for high-performance protein chromatography.
Co-reporter:Qianqian Wang, Linling Yu, Yan Sun
Journal of Chromatography A 2016 Volume 1443() pp:118-125
Publication Date(Web):22 April 2016
DOI:10.1016/j.chroma.2016.03.033
•Polymer-grafted anion exchangers of 14 chain densities and/or lengths were fabricated.•There was a specific ionic capacity that offered maximum protein binding capacity.•The highest static protein capacity reached 260 mg/mL, twice that of Q Sepharose.•The dynamic binding capacity of the resin was also much higher than Q Sepharose.To develop ion exchangers of high protein adsorption capacity, we have herein introduced atom transfer radical polymerization (ATRP) method to graft glycidyl methacrylate (GMA) onto Sepharose FF gel. GMA-grafted Sepharose FF resins of four grafting densities and different grafting chain lengths were obtained by adjusting reaction conditions. The epoxy groups on the grafted chains were functionalized by modification with diethylamine (DEA), leading to the fabrication of Sepharose-based anion exchangers of 14 different grafting densities and/or grafting chain lengths. The resins were first characterized for the effects of grafting density, chain length and ionic strength on pore sizes by inverse size exclusion chromatography. Then, the resins were evaluated by adsorption equilibria of bovine serum albumin (BSA) as a function of ionic capacity (IC) (chain length) at individual grafting densities. It was observed that at each grafting density there was a specific IC value (chain length) that offered the maximum equilibrium capacity. Of the resins with maximum values at individual grafting densities, the resin of the second grafting density with an IC value of 330 mmol/L (denoted as FF-Br2-pG-D330) showed the highest capacity, 264 mg/mL, about two times higher than that of the traditional ungrafted resin Q Sepharose FF (137 mg/mL). This resin also showed the most favorable uptake kinetics among the resins of similar IC values but different grafting densities, or of the same grafting density but different IC values. Effects of ionic strength showed that the capacities of FF-Br2-pG-D330 were much higher than Q Sepharose FF at a wide range of NaCl concentrations (0–200 mmol/L), and the uptake rates of the two resins were similar in the ionic strength range. Therefore, the dynamic binding capacity values of BSA on FF-Br2-pG-D330 were much higher than Q Sepharose FF as demonstrated at different residence times and ionic strengths. Taken together, the research has proved the success in the fabrication of high-capacity protein anion exchangers by grafting GMA onto Sepharose gel.
Co-reporter:Hu Liu, Xiaoyan Dong, Yan Sun
Journal of Chromatography A 2016 Volume 1429() pp:277-283
Publication Date(Web):15 January 2016
DOI:10.1016/j.chroma.2015.12.050
•High density metal chelators were prepared by grafting IDA monomer on silica NPs.•The nanoscale chelators facilitated refolding and purification of 6 × His-EGFP.•The chelators were found effective at protein concentrations up to 0.8 mg/mL.•Efficient refolding and purification were achieved at a very low chelator consumption.A series of highly charged nanoscale chelators were fabricated by grafting of poly(glycidyl methacrylate-iminodiacetic acid) (pGI) chains with iminodiacetic acid (IDA) chelating group on silica nanoparticles (SNPs) via atom transfer radical polymerization (ATRP). The nanoscale chelators, denoted as SNPs-pGI, possessed a nickel ion chelating capacity as high as 2800 μmol/g, 50 times higher than the IDA-modified Sepharose FF (IDA-Sepharose) resin reported in literature and offered a high affinity binding capacity for hexahistidine-tagged enhanced green fluorescence protein (6 × His-EGFP) after nickel ion loading. More importantly, the anionic SNPs-pGI of high charge densities displayed much better performance than IDA-Sepharose in facilitating the refolding of like-charged 6 × His-EGFP from inclusion bodies (IBs). For example, for 0.2 mg/mL 6 × His-EGFP IB refolding, addition of 6.2 μL/mL SNPs-pGI with the highest charge density led to a refolding yield of 90%, over 43% higher than that obtained with 460 μL/mL IDA-Sepharose. It is notable that the much higher efficiency of the nanoscale chelator was obtained with a chelator consumption corresponding to only 1.4% of IDA-Sepharose. Moreover, the highly charged SNPs-pGI could efficiently facilitate the refolding of 6 × His-EGFP at higher IB concentrations (0.4 and 0.8 mg/mL). After refolding, nickel ions addition led to the recovery of the refolded 6 × His-EGFP with high yield (80%), purity (96%) and enrichment ratio (1.8). All the results suggest that the SNPs-pGI of high charge densities were promising for cost-effective recovery of His-tagged proteins expressed as IBs with the integrative like-charge facilitated refolding and metal-chelate affinity purification strategy.
Co-reporter:Zhiqiang Jiang, Xiaoyan Dong, Hu Liu, Yongjian Wang, Lei Zhang, Yan Sun
Reactive and Functional Polymers 2016 Volume 104() pp:22-29
Publication Date(Web):July 2016
DOI:10.1016/j.reactfunctpolym.2016.04.019
Aggregation of amyloid β-protein (Aβ) is a major pathological hallmark of Alzheimer's disease. Curcumin has been recognized as an inhibitor of Aβ aggregation, but its low water solubility and bioavailability limits its clinical applications. In this work, we conjugated curcumin to hyaluronic acid and drastically improved its solubility and stability. We found that the conjugates self-assembled into nanosized hydrogels that inhibited Aβ fibrillogenesis and mitigated the amyloid cytotoxicity more efficiently than free curcumin. More importantly, we found that there was an optimal curcumin substitution degree, at which the hydrogel exhibited the strongest inhibitory effect. Based on these findings, a mechanistic model was proposed. It suggested that besides the inhibitory effect of the conjugated curcumin, HA provided three synergistic functions. The first is that curcumin encapsulation into nanogels protected cells from the toxicity of free curcumin. The second was an isolation effect of the hydrogel network, which hindered the interactions between Aβ molecules. The third was the counteraction of the hydrophobic binding between Aβ and the conjugated curcumin against the electrostatic repulsion between the like-charged Aβ and HA. The two opposite forces could stretch the conformation of Aβ monomers, slowing down the aggregation and/or leading to off-pathway aggregations. This work offered new insights into the development of more potent nanoparticles for inhibiting Aβ fibrillogenesis and cytotoxicity.
Co-reporter:Hongchen Liu, Baolong Xie, Xiaoyan Dong, Lei Zhang, Yongjian Wang, Fufeng Liu, Yan Sun
Reactive and Functional Polymers 2016 Volume 103() pp:108-116
Publication Date(Web):June 2016
DOI:10.1016/j.reactfunctpolym.2016.04.003
Self-assembly of amyloid β-protein (Aβ) is closely related to the pathogenesis of Alzheimer's disease (AD). Many studies suggest that polymeric nanoparticles (NPs) can inhibit Aβ fibrillogenesis depending on their electrostatic and hydrophobic properties, but the underlying molecular mechanism remains unknown. Herein, the inhibitory effect of NPs with equivalent content of hydrophobic groups but different surface negative charge densities on Aβ fibrillogenesis is examined. Firstly, the polymeric NPs of similar sizes were synthesized by copolymerizing equal proportion of N-isopropylacrylamide and different proportion of N-t-butylacrylamide and acrylic acid. Then, the inhibitory effects of these NPs on Aβ42 fibrillization and the corresponding cytotoxicity were investigated using thioflavin T fluorescent assay, transmission electron microscopy, dynamic light scattering analysis, and cell viability assay. It was found that these NPs showed remarkable inhibitory capability against Aβ42 fibrillogenesis and alleviated its cytotoxicity. The inhibitory capability significantly depended on the capacity of the negative surface charges carried by NPs with an increase-decrease trend. The best inhibitory efficiency was obtained at an optimal surface negative charge density. Based on the findings, a mechanistic model was proposed by considering the two interactions between Aβ42 and NPs, namely, hydrophobic binding and electrostatic repulsion. The model suggested that at an appropriate negative charge capacity, the two opposite forces could be well-balanced, and thus led to the stretching of Aβ42 molecules instead of the formation of a harmful β-sheet structure. The polymeric NPs of well-designed surface of proper hydrophobicity and negative charge density could thus significantly slow down the Aβ42 fibrillation and/or result in an off-pathway aggregation with reduced cytotoxicity.
Co-reporter:Neng Xiong, Xiao-Yan Dong, Jie Zheng, Fu-Feng Liu, and Yan Sun
ACS Applied Materials & Interfaces 2015 Volume 7(Issue 10) pp:5650
Publication Date(Web):February 20, 2015
DOI:10.1021/acsami.5b00915
Aggregation of amyloid β-protein (Aβ) into amyloid oligomers and fibrils is pathologically linked to Alzheimer’s disease (AD). Hence, the inhibition of Aβ aggregation is essential for the prevention and treatment of AD, but the development of potent agents capable of inhibiting Aβ fibrillogenesis has posed significant challenges. Herein, we designed Ac-LVFFARK-NH2 (LK7) by incorporating two positively charged residues, R and K, into the central hydrophobic fragment of Aβ17–21 (LVFFA) and examined its inhibitory effect on Aβ42 aggregation and cytotoxicity by extensive physical, biophysical, and biological analyses. LK7 was observed to inhibit Aβ42 fibrillogenesis in a dose-dependent manner, but its strong self-assembly characteristic also resulted in high cytotoxicity. In order to prevent the cytotoxicity that resulted from the self-assembly of LK7, the peptide was then conjugated to the surface of poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) to fabricate a nanosized inhibitor, LK7@PLGA-NPs. It was found that LK7@PLGA-NPs had little cytotoxicity because the self-assembly of the LK7 conjugated on the NPs was completely inhibited. Moreover, the NPs-based inhibitor showed remarkable inhibitory capability against Aβ42 aggregation and significantly alleviated its cytotoxicity at a low LK7@PLGA-NPs concentration of 20 μg/mL. At the same peptide concentration, free LK7 showed little inhibitory effect. It is considered that several synergetic effects contributed to the strong inhibitory ability of LK7@PLGA-NPs, including the enhanced interactions between Aβ42 and LK7@PLGA-NPs brought on by inhibiting LK7 self-assembly, restricting conformational changes of Aβ42, and thus redirecting Aβ42 aggregation into unstructured, off-pathway aggregates. The working mechanisms of the inhibitory effects of LK7 and LK7@PLGA-NPs on Aβ42 aggregation were proposed based on experimental observations. This work provides new insights into the design and development of potent NPs-based inhibitors against Aβ aggregation and cytotoxicity.Keywords: Alzheimer’s disease; amyloid β-protein; nanoparticle; peptide inhibitor; protein aggregation; self-assembly
Co-reporter:Linling Yu, Lin Zhang, Yan Sun
Journal of Chromatography A 2015 Volume 1382() pp:118-134
Publication Date(Web):20 February 2015
DOI:10.1016/j.chroma.2014.12.087
•Recent studies on protein behavior at chromatographic surfaces were reviewed.•Protein orientation at chromatographic surfaces and its implications were discussed.•Factors affecting protein unfolding/refolding at surfaces were summarized.•Surface transport of bound protein was elaborated with four mechanisms.•Discussion on the protein behavior offered insight into novel surface design.Chromatography is the key technology in protein purification as well as in protein refolding. Taking the scientific development and technological innovation of protein chromatography as the objective, this article is devoted to an overview of protein behavior at chromatographic surfaces, including protein orientation, conformational transitions (unfolding and refolding), and protein transport. Recent advances achieved by using molecular simulations as well as theoretical and experimental investigations are elaborated and discussed with emphasis on their implications to the rational design of novel chromatographic surfaces or materials and mobile phase conditions for the development of high-performance protein chromatography.
Co-reporter:Shi-Peng Tao, Jie Zheng, Yan Sun
Journal of Chromatography A 2015 Volume 1389() pp:104-111
Publication Date(Web):10 April 2015
DOI:10.1016/j.chroma.2015.02.051
•Zwitterionic polymer-grafted cryogel provided stronger surface hydrophilicity.•Effects of surface hydrophilicity and flow rate on SXC were investigated.•Physical properties of the columns remain unchanged after modification.•Protein capacity was 1.4–1.8 times increased by the zwitterionic polymer grafting.•The mechanism of enhanced protein retention on more hydrophilic surface was proposed.Cryogel monoliths with interconnected macropores (10–100 μm) and hydrophilic surfaces can be employed as chromatography media for protein retention in steric exclusion chromatography (SXC). SXC is based on the principle that the exclusion of polyethylene glycol (PEG) on both a hydrophilic chromatography surface and a protein favors their association, leading to the protein retention on the chromatography surface. Elution of the retained protein can be achieved by reducing PEG concentration. In this work, the surface of polyacrylamide-based cryogel monolith was modified by grafting zwitterionic poly(carboxybetaine methacrylate) (pCBMA), leading the increase in the surface hydrophilicity. Observation by scanning electron microscopy revealed the presence of the grafted pCBMA chain clusters on the cryogel surface, but pCBMA grafting did not result in the changes of the physical properties of the monolith column, and the columns maintained good recyclability in SXC. The effect of the surface grafting on the SXC behavior of γ-globulin was investigated in a wide flow rate range (0.6–12 cm/min). It was found that the dynamic retention capacity increased 1.4–1.8 times by the zwitterionic polymer grafting in the flow rate range of 1.5–12 cm/min. The mechanism of enhanced protein retention on the zwitterionic polymer-grafted surface was proposed. The research proved that zwitterionic polymer modification was promising for the development of new materials for SXC applications.
Co-reporter:Hu Liu, Xiaoyan Dong, and Yan Sun
Langmuir 2015 Volume 31(Issue 2) pp:655-658
Publication Date(Web):January 6, 2015
DOI:10.1021/la5040454
Silica nanoparticles (SNPs) were sequentially modified with poly(ethylenimine) (PEI) and 2-diethylaminoethyl chloride (DEAE) to prepare a series of positively charged SNPs-PEI and SNPs-PEI-DEAE. The sequential double-modification strategy produced a charge density as high as 1740 μmol/g (4524 μmol/mL), which offered a very high adsorption capacity for bovine serum albumin (314 mg/g). Most importantly, the highly charged SNPs-PEI and SNPs-PEI-DEAE could efficiently facilitate the refolding of like-charged protein at extremely low utilization. For instance, in the refolding of 1 mg/mL lysozyme, the refolding yield reached 75% with only 3.3 μL/mL SNPs-PEI-DEAE. The bead consumption was reduced by nearly 96% as compared to that of the charged microspheres used previously to reach a similar yield. The results proved that the polyelectrolyte-modified SNPs were promising for applications in facilitating like-charged protein refolding, and the research opened up a new way for biotechnology applications of highly charged nanoparticles.
Co-reporter:Baolong Xie, Xiaoyan Dong, Yongjian Wang, and Yan Sun
Langmuir 2015 Volume 31(Issue 26) pp:7374-7380
Publication Date(Web):June 12, 2015
DOI:10.1021/acs.langmuir.5b01108
Fibrillogenesis of amyloid β-proteins (Aβ) mediated by transition-metal ions such as Zn2+ in neuronal cells plays a causative role in Alzheimer’s disease. Hence, it is highly desired to design multifunctional agents capable of inhibiting Aβ aggregation and modulating metal–Aβ species. In this study, we fabricated acidulated human serum albumin (A-HSA) as a multifunctional agent for binding Zn2+ and modulating Zn2+-mediated Aβ fibrillogenesis and cytotoxicity. On average, 19.5 diglycolic anhydrides were modified onto the surface of human serum albumin (HSA). It was confirmed that A-HSA kept the stability and biocompatibility of native HSA. Moreover, it could inhibit Aβ42 fibrillogenesis and change the pathway of Zn2+-mediated Aβ42 aggregation, as demonstrated by extensive biophysical assays. In addition, upon incubation with A-HSA, the cytotoxicity presented by Zn2+–Aβ42 aggregates was significantly mitigated in living cells. The results showed that A-HSA had much stronger inhibitory effect on Zn2+-mediated Aβ42 fibrillogenesis and cytotoxicity than equimolar HSA. Isothermal titration calorimetry and stopped-flow fluorescence measurements were then performed to investigate the working mechanism of A-HSA. The studies showed that the A-HSA surface, with more negative charges, not only had stronger affinity for Zn2+ but also might decrease the binding affinity of Aβ42 for Zn2+. Moreover, hydrophobic binding and electrostatic repulsion could work simultaneously on the bound Aβ42 on the A-HSA surface. As a result, Aβ42 conformations could be stretched, which avoided the formation of toxic Zn2+–Aβ42 aggregates. The research thus revealed that A-HSA is a multifunctional agent capable of altering the pathway of Zn2+-mediated Aβ42 aggregation and greatly mitigating the amyloid cytotoxicity.
Co-reporter:Na Liu, Ziyuan Wang, Xiaoguang (Margaret) Liu, Linling Yu, Yan Sun
Journal of Chromatography A 2014 Volume 1372() pp:157-165
Publication Date(Web):12 December 2014
DOI:10.1016/j.chroma.2014.10.108
We have previously studied poly(ethylenimine) (PEI)-grafted Sepharose FF resins for ion-exchange chromatography of bovine serum albumin (BSA), and found the presence of a critical ionic capacity (cIC, 600 mmol/L for BSA), above which both BSA adsorption capacity and uptake rates increased drastically. To extend the application of PEI-grafted resins, we have herein proposed to develop mixed-mode chromatographic (MMC) resins by modifying the grafted PEI chains with hydrophobic benzoyl groups. Three PEI-grafted resins with IC values from 329 to 701 mmol/L (FF-PEI-L330, FF-PEI-L520 and FF-PEI-L700) were modified with benzoic acid. It was found that there was a maximum benzoyl density (BD) that could be reached for each resin, at which the average value of BD/residual IC was 1.2. The effect of BD (120–400 mmol/L) on BSA adsorption (at pH 8.0) and elution (at pH 3.0) was first explored with FF-PEI-L700-derived resins. It was observed that both protein binding capacity and recovery increased with increasing BD, indicating that high BD was beneficial in protein adsorption. However, the elution of bound BSA with an acidic buffer (pH 3.0) was incomplete. It was hypothesized that the PEI chains, a pH-dependent cationic polyelectrolyte, formed a collapsed layer at the protein binding condition (high ionic strength, IS), while they exhibited extended spatial structures at elution (low pH and IS). These PEI chain structure transitions made the pores change from an opening state at the loading condition to a blocked state at the elution condition. The pore blocking was regarded as a chain-aroused-steric-hindrance (CaSH) effect. Thus, FF-PEI-L700 was not suitable for fabricating MMC resins due to its high chain density. Then, the effect of PEI density (the initial IC values of 330–700 mmol/L) at the maximum BD values was investigated. Consequently, complete BSA recovery at pH 3.0 was obtained on the resin with an initial IC of 330 mmol/L and a BD of 160 mmol/L (B160-PEI330). The result indicates that the CaSH effect could be ignored at the low PEI chain density (IC = 329 mmol/L). Moreover, selective elution of γ-globulin could be achieved at pH 4.0 on the B160-PEI330 column, indicating the possibility of antibody purification from a mixture containing albumin by manipulating elution conditions. Finally, adsorption equilibria and uptake kinetics onto B160-PEI330 showed favorable binding properties for different proteins at a wide range of IS, indicating its usefulness as an MMC adsorbent.
Co-reporter:Shi-Peng Tao, Chuan Wang, Yan Sun
Journal of Chromatography A 2014 Volume 1359() pp:76-83
Publication Date(Web):12 September 2014
DOI:10.1016/j.chroma.2014.07.019
•Nanoparticles (NPs)-coated cryogel provided larger specific surface area.•Cryogel ion-exchanger was fabricated by NPs-coating and double modifications.•Protein capacity was 4.2 times increased by the coating and double modifications.•Column efficiency and capacity decreased only slightly from 0.6 to 12 cm/min.A novel composite cryogel monolith was developed by coating poly(glycidyl methacrylate) nanoparticles (NPs) onto the pore wall surface of poly(acrylamide) cryogel. The NPs-coated column was double-modified with poly(ethylenimine) (PEI) and diethylaminoethyl in sequence. Scanning electron microscopy revealed the dense coating of the NPs on the cryogel surface, but the NPs-coating did not result in distinct changes of the column porosity and permeability. The rough pore wall surface and extended polymer chains offered more binding sites, so the dynamic binding capacity of the composite cryogel bed for bovine serum albumin reached 11.7 mg/mL bed volume at a flow rate of 6 cm/min, which was 4.2 times higher than that of the cryogel bed modified with PEI without coating NPs (2.8 mg/mL). The binding capacity as well as column efficiency decreased only slightly with increasing flow rate from 0.6 to 12 cm/min. The results indicated that the strategy of NPs-coating incorporating with double ion-exchanger modifications is promising for enhancing cryogel capacities, and the novel material would be useful for high-speed protein chromatography.
Co-reporter:Wei-Wei Zhao, Fu-Feng Liu, Qing-Hong Shi, Yan Sun
Journal of Chromatography A 2014 Volume 1359() pp:100-111
Publication Date(Web):12 September 2014
DOI:10.1016/j.chroma.2014.07.023
•Affinity chromatography of antibody based on three octapeptide ligands was reported.•The ligands bound Fc (not Fab) and the binding sites were similar to those for SpA.•Adsorption equilibria of hIgG were compared for the different affinity resins.•hIgG and mAb purifications were achieved at high purities and recovery yields.•MD simulations revealed molecular interactions of the octapeptides and Fc fragment.In an earlier work, we have developed a biomimetic design strategy based on the human IgG (hIgG)–Protein A interactions and identified an affinity ligand for hIgG, FYWHCLDE, which ranked top one in a pool of 14 potential candidates. Herein, two more octapeptides, FYCHWALE and FYCHTIDE, were identified, and the binding and purification of hIgG on the affinity columns packed with the three octapeptide-modified Sepharose gels were extensively studied and compared to find more effective octapeptide-based affinity ligands. It was found that all the three ligands bound hIgG and Fc fragment but barely bound Fab fragment, and the binding to hIgG and Fc was mainly by electrostatic interactions. The optimum binding pH values for the three ligands were different from each other, but kept in the range of 5.0–6.0. Ligand binding competition revealed that the binding sites on hIgG for the three octapeptides were similar to those for Protein A. Adsorption isotherms revealed that hIgG binding capacity was in the range of 64–104 mg/mL drained gel in the order of FYWHCLDE > FYCHWALE > FYCHTIDE. Then, purifications of hIgG and human monoclonal antibody from human serum and cell culture supernatant, respectively, were achieved with the three affinity columns at high purities and recovery yields. Finally, the molecular basis for the binding affinity of the peptides for the Fc fragment of hIgG was elucidated by molecular dynamics simulations.
Co-reporter:Wei-Wei Zhao, Qing-Hong Shi, Yan Sun
Journal of Chromatography A 2014 Volume 1355() pp:107-114
Publication Date(Web):15 August 2014
DOI:10.1016/j.chroma.2014.05.083
•FYWHCLDE-based affinity chromatography for antibody purification was reported.•Effects of ligand density on adsorption equilibria and kinetics were investigated.•hIgG and mAb purifications were achieved at high purities and recovery yields.•Robustness of the affinity column was demonstrated by 20 recycled uses.This work reports the development of an octapeptide-based affinity adsorbent for the purification of human IgG (hIgG) and monoclonal antibody (mAb). The octapeptide was FYWHCLDE selected earlier by the biomimetic design of affinity peptide ligands for hIgG. The ligand was coupled to Sepharose gel at four densities from 10.4 to 31.0 μmol/mL, and the effect of peptide density on the adsorption of hIgG and bovine serum albumin (BSA) was first investigated. The binding capacity of hIgG increased from 104.2 to 176.4 mg/mL within the ligand density range, and the binding affinity (dissociation constant) kept at 2.4–3.7 μM. Batch adsorption revealed that the selectivity of FYWHCLDE-Sepharose for IgG was 30–40 times over BSA. The effective pore diffusivity of IgG decreased somewhat with increasing ligand density, but the dynamic binding capacity at 10% breakthrough, measured by using 10-fold diluted human serum as feedstock, doubled with increasing ligand density from 10.4 to 31.0 μmol/mL due to the remarkable increase of static binding capacity. By using the affinity column with a ligand density of 23.9 μmol/mL, hIgG and humanized mAb purifications from human serum and cell culture supernatant, respectively, were achieved at high purities and recovery yields. Finally, the robustness of the peptide gel was demonstrated by recycled use of the affinity column in 20 breakthrough cycles
Co-reporter:Yanying Li, Yan Sun
Journal of Chromatography A 2014 Volume 1373() pp:97-105
Publication Date(Web):19 December 2014
DOI:10.1016/j.chroma.2014.11.017
•Poly(4-vinylpyridine) was coupled to Sepharose gel for use as a protein ligand.•Both electrostatic and hydrophobic interactions worked on protein adsorption.•Electrostatic interaction played the dominant role in protein adsorption.•Bound protein could be recovered by elution with a buffer of pH 4.0 or 4.5.Poly(4-vinylpyridine) (P4VP) was proposed for use as a polymeric ligand of mixed-mode chromatography (MMC) of proteins. P4VP has linear hydrophobic chains with ionizable pyridyl groups in its backbone. The polymer was coupled onto Sepharose FF gel at a pyridyl group density of 190 μmol/mL (FF-P4VP-190) by the substitution reaction of pyridyl amines with brominated Sepharose gel. Thereby the immobilized ligand possesses the intrinsic hydrophobic nature as well as the newly obtained electrostatic interaction properties endowed from the substituted positively charged pyridyl amines. The pore size distribution was measured by inverse size exclusion chromatography, and the results revealed that P4VP formed a three-dimensional layer on the matrix surface with a maximum layer depth of 4.2 nm. The adsorption isotherms of γ-globulin and bovine serum albumin (BSA) to FF-P4VP-190 were determined under varying pH values and salt concentrations to provide insights into the adsorption properties of the medium. It was found that the adsorption capacity of γ-globulin and BSA both presented an increase with pH increasing from 8.0 to 9.0. Moreover, FF-P4VP-190 exhibited stronger adsorption for BSA than γ-globulin. The higher affinity for BSA might be attributed to its more net negative charges. Protein adsorption capacities to FF-P4VP-190 decreased with increasing NaCl concentration, but still manifested moderate levels at high salt concentration such as 75 mg/mL for γ-globulin and 14 mg/mL for BSA at 0.5 mol/L NaCl and pH 9.0. The capacity decreases with increasing ionic strength, indicating the dominant role of electrostatic interactions, while the moderate capacity values at 0.5 mol/L NaCl confirmed the presence of salt-tolerant feature of FF-P4VP-190, making it function as an MMC material. Column chromatography was conducted to investigate protein elution behavior. Efficient protein recovery was achieved at mild elution conditions such as pH 4.0 for γ-globulin and pH 4.5 for BSA. The results indicate that the P4VP-based adsorbent would provide new possibilities for protein purification by MMC.
Co-reporter:Wei-Wei Zhao, Qing-Hong Shi, Yan Sun
Journal of Chromatography A 2014 Volume 1369() pp:64-72
Publication Date(Web):21 November 2014
DOI:10.1016/j.chroma.2014.09.083
•Dual-ligand affinity systems for antibody adsorption and purification were reported.•Synergistic effect of dual ligands were found and elucidated by MD simulations.•The effect increased with increasing total ligand density in a dual-ligand system.•The effect resulted in a proper molar ratio of ligands for the highest affinity.•hIgG and mAb purifications were achieved at higher purities and recovery yields.This work reports the development of affinity systems with dual octapeptide ligands for affinity adsorption and purification of human IgG (hIgG) and monoclonal antibody (mAb). The three octapeptide ligands, FYWHCLDE (1), FYCHWALE (2), and FYCHTIDE (3), identified earlier by the biomimetic design strategy were used; any two of the three were mixed and coupled to Sepharose gel, leading to the formation of three dual-ligand affinity systems. Research emphasis was first placed on hIgG adsorption isotherms and the results were compared to the three single-ligand affinity systems. It was found that there was synergistic effect of the two peptide ligands in a dual-ligand system, so the affinity of a dual-ligand resin for hIgG was higher than those of its counterparts, single-ligand resins. Of the three dual-ligand systems, the FYWHCLDE (1)–FYCHTIDE (3) resin showed the highest affinity, so it was selected for investigating the effects of ligand density and molar ratio on hIgG adsorption equilibrium. It was found that the synergistic effect increased with increasing the total ligand density of the two peptides in the dual-ligand affinity system. Moreover, the FYWHCLDE (1)–FYCHTIDE (3) system at a molar ratio of 2:1 displayed the highest affinity for hIgG (0.69 μM at a total ligand density of 31.1 μmol/mL), indicating that the synergistic effect reached the maximum at this ratio. This dual-ligand affinity column was then used for the purification of hIgG and mAb by affinity chromatography, resulting in over 95% pure hIgG and mAb at recovery yield over 90%. Molecular docking of the two peptides to the Fc fragment simultaneously showed that FYWHCLDE (1) stood still but FYCHTIDE (3) shifted aside the CH2CH3 inter-domain. Molecular dynamics simulation of the binding process of the two octapeptides to Fc revealed that both the peptide ligands kept stable interactions with Fc. The synergistic effect of the dual-ligand affinity system was thus elucidated by the molecular simulations.
Co-reporter:Na Liu, Lin-Ling Yu, Yan Sun
Journal of Chromatography A 2014 Volume 1362() pp:218-224
Publication Date(Web):3 October 2014
DOI:10.1016/j.chroma.2014.08.052
•Dynamic BSA adsorption and elution were studied on PEI-Sepharose columns.•FF-PEI-L680 kept high dynamic capacities at wide residence times and ionic strengths.•Symmetrical elution peaks were obtained for the PEI resins as normal ungrafted resins.•The ionic strength for elution in linear gradient elution increased with PEI density.We have previously investigated bovine serum albumin (BSA) uptake to poly(ethylenimine) (PEI)-grafted Sepharose FF. It was found that there was a critical ionic capacity (cIC; 600 mmol/L) for BSA, above which the protein adsorption capacity and uptake kinetics increased drastically. In this work, two poly(ethylenimine) (PEI)-grafted resins with IC values of 271 mmol/L (FF-PEI-L270) and 683 mmol/L (FF-PEI-L680), which were below and above the cIC, respectively, were chosen to investigate the breakthrough and linear gradient elution (LGE) behaviors of BSA. Commercially available anion exchanger, Q Sepharose FF, was used for comparison. The DBC values of FF-PEI-L680 were much higher in the entire residence time range (2−10 min) than the other two resins due to its high static adsorption capacity and uptake kinetics. At a residence time of 5.0 min, the DBC of FF-PEI-L680 (104 mg/mL) was about seven times that of FF-PEI-L270 and three times that of Q Sepharose FF. A rise–fall trend of the DBCs with increasing ionic strength (IS) was found for all the three resins studied, indicating the presence of electrostatic exclusion for protein uptake at low IS. With increasing NaCl concentration from 20 to 200 mmol/L, FF-PEI-L680 kept very high DBC values (64−114 mg/mL). In addition, FF-PEI-L270 showed more favorable adsorption properties than Q Sepharose FF at 100–300 mmol/L NaCl. These results proved that the three-dimensional grafting ion exchange layer on the PEI resins enhanced their tolerance to IS. In the study of LGE, the three resins showed similar elution behaviors and no distinct peak tailings were observed. The salt concentrations at the elution peaks (IR) were in the order of FF-PEI-L680 > FF-PEI-L270 > Q Sepharose FF, indicating that the elution for the PEI resins needed higher salt concentrations, which was also an appearance of the salt-tolerant feature of the PEI resins. When protein loading amount was increased to the value equivalent to the DBC at 10% breakthrough, the adsorbed BSA could be eluted at lower salt concentrations. The chromatographic study has provided new insights into the practical application of the PEI-based anion exchangers.
Co-reporter:Xiao-Yan Dong, Ran Chen, Chun-Yan Yang, Yan Sun
Journal of Chromatography A 2014 Volume 1347() pp:49-55
Publication Date(Web):20 June 2014
DOI:10.1016/j.chroma.2014.04.055
•Sequential grafting and charge modification enhanced sulfonate density to 793 μmol/g.•Enhanced EGFP refolding and purification were realized with the highly charged beads.•52% of contaminant proteins was removed by adsorption to the charged beads.•Reusability of the charged beads was demonstrated in recycled protein refolding.We have previously found that addition of like-charged media in a refolding solution can greatly enhance the refolding of pure proteins by suppressing protein aggregation. Herein, negatively charged mono-sized microspheres with sulfonic groups were fabricated to explore the facilitating effect of like-charged media on the refolding of enhanced green fluorescent protein (EGFP) expressed as inclusion bodies (IBs). A sequential polymer-tentacle grafting and sulfonate modification strategy was developed to increase the charge density of mono-sized poly(glycidyl methacrylate) (pGMA) beads (2.4 μm). Namely, GMA was first grafted onto the beads by grafting polymerization to form poly(GMA) tentacles on the pGMA beads, and then the epoxy groups on the tentacles were converted into sulfonic groups by modification with sodium sulfite. By this fabrication strategy, the charge density of the beads reached 793 μmol/g, about 2.8 times higher than that modified without prior grafting of the pGMA beads (285 μmol/g). The negatively charged beads of different charge densities were used for facilitating the refolding of like-charged EGFP from IBs. The refolding yield as well as refolding rate increased with increasing charge density. The anti-aggregation effects of urea and like-charged microspheres were synergetic. In addition, partial purification of EGFP was achieved because the ion-exchange adsorption led to 52% removal of positively charged contaminant proteins in the refolded solution. Finally, reusability of the tentacle beads was demonstrated by repetitive EGFP refolding and recovery cycles.
Co-reporter:Hu Liu, Wen-Jie Du, Xiao-Yan Dong, Yan Sun
Journal of Chromatography A 2014 Volume 1344() pp:59-65
Publication Date(Web):30 May 2014
DOI:10.1016/j.chroma.2014.04.006
•An integrative method of protein refolding and metal-chelate capture was proposed.•Iminodiacetic acid modified resins facilitate refolding of like-charged His-tagged protein.•Addition of nickel ions leads to the affinity capture of the His-tagged protein.•Facilitated refolding and purification of the His-tagged protein are realized.This work proposed an integrative method of protein refolding and purification by like-charged resin facilitated refolding and metal-chelate affinity adsorption. Hexahistidine-tagged enhanced green fluorescence protein (EGFP) was overexpressed in Escherichia coli as inclusion bodies (IBs), and then the protein was refolded and purified from urea-solubilized IBs by this method. A metal-chelating resin was fabricated by coupling iminodiacetic acid (IDA) to agarose gel (Sepharose FF). The anionic resin was used to facilitate the refolding of like-charged EGFP from IBs. After refolding, nickel ions were introduced for the affinity purification of the target protein by metal-chelating adsorption. It was found that the resin was effective in facilitating EGFP refolding. For 0.1 mg/mL EGFP IBs refolding, the fluorescence recovery (FR) by direct dilution was only 64%; addition of only 0.05 g/mL resin increased the FR to over 90%. Moreover, the FR increased with increasing resin concentration. Owning to the shielding effect of the oppositely charged impurities embedded in IBs on the surface charges of the IDA resin, more resin particles were required to exert an aggregation inhibition effect in the IBs protein refolding. Additionally, compared with direct-dilution refolding, inclusion of like-charged resins not only offered an enhanced FR of EGFP, but also bound some opposite-charged contaminant proteins, leading to a preliminary purification effect. Afterwards, the refolded EGFP was recovered by metal-chelating adsorption at an FR of 85% and purity of 93%. This work has thus extended the like-charge facilitated protein refolding strategy to the integrative protein refolding and purification.
Co-reporter:Yan Hong, Na Liu, Wei Wei, Lin-Ling Yu, Guanghui Ma, Yan Sun
Journal of Chromatography A 2014 Volume 1342() pp:30-36
Publication Date(Web):16 May 2014
DOI:10.1016/j.chroma.2014.03.036
•γ-Globulin adsorption to PEI-Sepharose was studied and compared with BSA.•γ-Globulin capacity increased with ionic capacity (IC) at IC < 560 mmol/L.•The IC where γ-globulin uptake rate started to hop was earlier than that of BSA.•γ-Globulin uptake rate at IC = 680 mmol/L was four times of that at IC < 460 mmol/L.•Both capacity and uptake rate of γ-globulin decreased drastically at IC > 680 mmol/L.Previously, we studied bovine serum albumin (BSA) uptake to poly(ethylenimine) (PEI)-grafted Sepharose resins, and an ionic capacity (IC) range (600–740 mmol/L) for steep increases of both protein capacity (qm) and effective pore diffusion coefficient (De) was found. In this work, seven PEI-grafted Sepharose FF resins at IC range of 270–1030 mmol/L were synthesized to investigate the effect of protein properties on the adsorption and uptake kinetics using BSA and γ-globulin as two model proteins. For BSA, the change trends of qm and De values with IC were well consistent with the previous results. For γ-globulin, the qm values increased slowly till reaching a maximum value at IC = 560 mmol/L and then decreased rapidly at IC > 560 mol/L. The De values nearly kept unchanged at low ICs (IC < 460 mmol/L), and increased steeply at IC > 460 mmol/L till reaching a maximum at 680 mmol/L (De/D0 = 0.48 ± 0.01). After that increase, the De values for γ-globulin dropped quickly at IC > 680 mol/L, which was not observed for BSA. It is interesting to note that in the narrow IC range of 460–680 mmol/L, the De values of γ-globulin increased dramatically for more than four folds. Moreover, it is notable that the IC range where the hopping of De values occurred for γ-globulin was earlier than that for BSA (460 vs. 560 mmol/L). The earlier hopping of γ-globulin uptake rate was attributed to its larger size and less net charge, which facilitated the happenings of the “chain delivery” effect. The quick drops of both qm and De values for γ-globulin at IC > 680 mmol/L were considered due to its large size, which led to the significant decrease of its effective pore volume. The results indicate that both PEI layer and protein size played important roles in protein adsorption to PEI-grafted resins, and further prove the “chain delivery” effect did contributed significantly to the uptake rate hopping in the PEI-grafted resins. This work could also help the design and selection of resins based on protein characteristics and benefit optimization of practical chromatographic processes for therapeutic proteins with PEI-grafted anion exchangers.
Co-reporter:Chuan Wang, Shu Bai, Shi-Peng Tao, Yan Sun
Journal of Chromatography A 2014 Volume 1333() pp:54-59
Publication Date(Web):14 March 2014
DOI:10.1016/j.chroma.2014.01.059
•Steric exclusion chromatography was evaluated on cryogel monolith column.•The retention capacity of the cryogel column for γ-globulin reached 20 mg/mL.•Compact packing of protein precipitates on the cryogel surface was revealed by SEM.•Primary separation of serum proteins was achieved with concentrated γ-globulin.Steric exclusion chromatography (SXC) is a new mode of protein chromatography, in which large proteins are retained on hydrophilic stationary phase surface due to the steric exclusion of polyethylene glycol (PEG) in the mobile phase, and thereafter the retained proteins can be eluted by reducing PEG concentration. In this work, SXC was evaluated on a polyacrylamide cryogel monolith. Microscopic observation of γ-globulin precipitates on the gel surface in SXC was reported for the first time. Due to the compact packing of protein precipitates on the stationary phase surface, the dynamic retention capacity of the cryogel monolith for γ-globulin reached 20 mg/mL bed volume, much higher than those of cryogel beds in adsorption-based chromatography. The effect of molecular weight and concentration of PEG, solution pH and salt concentration on protein retention capacity was in agreement with the earlier work on SXC. Because the cryogel monoliths with interconnected macropores (10–100 μm) allow much easy flow-through of viscous PEG buffer, the SXC can be operated at low back pressure. Hence, the cryogel monoliths are more suitable for SXC than other monoliths of narrow pores reported previously. In the separation of bovine serum proteins, albumin was recovered in the breakthrough fraction with high purity, and globulin was over eight times concentrated in the elution pool. This work has, thus, demonstrated the rapid serum protein separation and concentration by SXC on the cryogel monolith columns.
Co-reporter:Shuo Wang, Shu Bai, Xiao-Yan Dong, Yan Sun
Separation and Purification Technology 2014 Volume 133() pp:149-154
Publication Date(Web):8 September 2014
DOI:10.1016/j.seppur.2014.07.005
•Nickel-chelate aqueous two-phase micellar system (ATPMS) was constructed.•EGFP partitioning behavior in the affinity partitioning system was investigated.•Partition coefficient of EGFP was remarkably enhanced by nickel-chelated Triton X114.•EGFP was purified directly from cell lysate using the affinity-based ATPMS.Aqueous two-phase micellar system (ATPMS), an alternative to chromatography, has been considered as a promising liquid–liquid extraction technique for biomolecule purification. To improve the specificity of ATPMSs, a nickel-chelated surfactant (TX-Ni) has been fabricated. The affinity-based ATPMS formed by Triton X-114 (TX) and TX-Ni was characterized for the purification of recombinant hexahistidine-tagged enhanced green fluorescent protein (EGFP). The stability of EGFP in the ATPMS was first confirmed. Then, the affinity binding of EGFP to TX-Ni was proved by investigation of the partitioning behavior. Thereafter, EGFP was extracted directly from cell lysate by the Ni(II)-chelated ATPMS. It was found that, more impurities were removed to the micelle-poor phase with increasing NaCl concentration, and the increase of TX-Ni gave rise to a recovery of EGFP over 90%. Finally, ethylenediaminetetraacetic acid (EDTA) was used to back-extract EGFP, presenting a total recovery yield of 83% with a purity of 70%. The results indicate that the affinity-based ATPMS is promising for the primary separation of histidine-rich proteins.
Co-reporter:Lin Zhang, Chao Zhang, and Yan Sun
Langmuir 2014 Volume 30(Issue 16) pp:4734-4742
Publication Date(Web):2017-2-22
DOI:10.1021/la4046012
Platelet adhesion on collagen mediated by integrin α2β1 has been proven important in arterial thrombus formation, leading to an exigent demand on development of potent inhibitors for the integrin α2β1-collagen binding. In the present study, a biomimetic design strategy of platelet adhesion inhibitors was established, based on the affinity binding model of integrin proposed in part I. First, a heptapeptide library containing 8000 candidates was designed to functionally mimic the binding motif of integrin α2β1. Then, each heptapeptide in the library was docked onto a collagen molecule for the assessment of its affinity, followed by a screening based on its structure similarity to the original structure in the affinity binding model. Eight candidates were then selected for further screening by molecular dynamics (MD) simulations. Thereafter, three candidates chosen from MD simulations were separately added into the physiological saline containing separated integrin and collagen, to check their abilities for blocking the integrin–collagen interaction using MD simulations. Of these three candidates, significant inhibition was observed in the presence of LWWNSYY. Finally, the binding affinity of LWWNSYY for collagen was demonstrated by isothermal titration calorimetry. Moreover, significant inhibition of platelet adhesion in the presence of LWWNSYY has been experimentally validated. This work has thus developed an effective strategy for the biomimetic design of peptide-based platelet adhesion inhibitors.
Co-reporter:Lin Zhang and Yan Sun
Langmuir 2014 Volume 30(Issue 16) pp:4725-4733
Publication Date(Web):2017-2-22
DOI:10.1021/la404599s
Platelet adhesion on a collagen surface through integrin α2β1 has been proven to be significant for the formation of arterial thrombus. However, the molecular determinants mediating the integrin–collagen complex remain unclear. In the present study, the dynamics of integrin–collagen binding and molecular interactions were investigated using molecular dynamics (MD) simulations and molecular mechanics–Poisson–Boltzmann surface area (MM-PBSA) analysis. Hydrophobic interaction is identified as the major driving force for the formation of the integrin–collagen complex. On the basis of the MD simulation and MM-PBSA results, an affinity binding model (ABM) of integrin for collagen is constructed; it is composed of five residues, including Y157, N154, S155, R288, and L220. The ABM has been proven to capture the major binding motif contributing 84.8% of the total binding free energy. On the basis of the ABM, we expect to establish a biomimetic design strategy of platelet adhesion inhibitors, which would be beneficial for the development of potent peptide-based drugs for thrombotic diseases.
Co-reporter:Yanying Li, Xiaodan Liu, Xiaoyan Dong, Lin Zhang, and Yan Sun
Langmuir 2014 Volume 30(Issue 28) pp:8500-8508
Publication Date(Web):June 29, 2014
DOI:10.1021/la5017438
Virus-like particle (VLP) of murine polyomavirus (MPV) is a T = 7d icosahedral capsid that self-assembles from 72 capsomeres (Caps), each of which is a pentamer of major coat protein VP1. VLP has great potential in vaccinology, gene therapy, drug delivery, and materials science. However, its application is hindered by high cost downstream processes, leading to an urgent demand of a highly efficient affinity ligand for the separation and purification of Cap by affinity chromatography. Herein a biomimetic design strategy of an affinity peptide ligand of Cap has been developed on the basis of the binding structure of the C-terminus of minor coat protein (VP2-C) on the inner surface of Cap. The molecular interactions between VP2-C and Cap were first examined using all-atom molecular dynamics (MD) simulations coupled with the molecular mechanics/Poisson–Boltzmann surface area (MM/PBSA) method, where V283, P285, D286, W287, L289, and Y296 of VP2-C were identified as the hot spots. An affinity peptide library (DWXLXLXY, X denotes arbitrary amino acids except cysteine) was then constructed for virtual screening sequently by docking with AUTODOCK VINA, binding structure comparison, and final docking with ROSETTA FlexPepDock. Ten peptide candidates were selected and further confirmed by MD simulations and MM/PBSA, where DWDLRLLY was found to have the highest affinity to Cap. In DWDLRLLY, six residues are favorable for the binding, including W2, L4, L6 and Y8 inheriting from VP2-C, and R5 and L7 selected in the virtual screening. This confirms the high efficiency and accuracy of the biomimetic design strategy. DWDLRLLY was then experimentally validated by a one-step purification of Cap from crude cell lysate using affinity chromatography with the octapeptide immobilized on Sepharose gel. The purified Caps were observed to self-assemble into VLP with consistent structure of authentic MPV.
Co-reporter:Baolong Xie, Xi Li, Xiao-Yan Dong, and Yan Sun
Langmuir 2014 Volume 30(Issue 32) pp:9789-9796
Publication Date(Web):August 1, 2014
DOI:10.1021/la5025197
Alzheimer’s disease (AD) is the most prevalent form of dementia, and aggregation of amyloid β-proteins (Aβ) into soluble oligomers and fibrils has been implicated in the pathogenesis of AD. Herein we developed acidulated serum albumin for the inhibition of Aβ42 fibrillogenesis. Bovine serum albumin (BSA) was modified with diglycolic anhydride, leading to the coupling of 14.5 more negative charges (carboxyl groups) on average on each protein surface. The acidulated BSA (A-BSA) was characterized and confirmed to keep the tertiary structure and stability of BSA. Extensive biophysical and biological analyses showed that A-BSA significantly inhibited Aβ42 fibrillogenesis and mitigated amyloid cytotoxicity. As compared to the Aβ42-treated group (cell viability, 50%), the cell viability increased to 88% by the addition of equimolar A-BSA. The inhibitory effect was remarkably higher than that of BSA at the same concentration. On the basis of the experimental findings, a mechanistic model was proposed. The model considers that Aβ42 is bound to the A-BSA surface by hydrophobic interactions, but the widely distributed negative charges on the A-BSA surface give rise to electrostatic repulsions to the bound Aβ42 that is also negatively charged. The two well-balanced opposite forces make Aβ42 adopt extended conformations instead of the β-sheet structure that is necessary for the on-pathway fibrillogenesis, even when the protein is released off the surface. Thus, A-BSA greatly slows down the fibrillation and changes the fibrillogenesis pathway, leading to the formation of less toxic aggregates. The findings and the mechanistic model offer new insights into the development of more potent inhibitors of Aβ fibrillogenesis and cytotoxicity.
Co-reporter:Lin-Ling Yu, Yan Sun
Journal of Chromatography A 2013 Volume 1305() pp:85-93
Publication Date(Web):30 August 2013
DOI:10.1016/j.chroma.2013.07.016
•Protein capacity on PEI-Sepharose was less sensitive to salt than traditional resins.•Sensitivity of protein uptake kinetics to salt was positively related to PEI density.•Protein uptake rates to low PEI-density resins were slower than non-grafting resins.•Uptake capacity and rate at high PEI densities showed maxima at certain salt concentration.•Effective diffusivity could be over 1.6 times of free diffusivity.In Part I of this work, we have studied the effect of ionic capacity (IC) on bovine serum albumin (BSA) adsorption equilibria and kinetics to poly(ethylenimine) (PEI)-grafted Sepharose FF, and found a critical IC (cIC, 600 mmol/L), above which both protein capacity and uptake rate increased drastically. In this work, five PEI-Sepharose FF resins of typical ICs reported earlier were selected to explore the effect of ionic strength (IS) on the adsorption equilibria and kinetics of BSA. Commercially available DEAE (IC = 160 mmol/L) and Q Sepharose FF (IC = 269 mmol/L) resins were used for comparisons. It is found that at similar ionic capacities, protein adsorption capacities on both the PEI-Sepharose FF resins and the commercial resins decreased with increasing IS, but on the capacity sensitivity to salt concentration, the former was lower than the latter. In addition, the effective diffusivities (De) of the former were smaller than the latter in the entire IS range studied. The low IS sensitivity of adsorption capacity of the PEI-Sepharose FF resins could be interpreted by the increase of pore accessibility with increasing IS; the smaller De values in the PEI-Sepharose FF resins were considered due to the lack of surface diffusion in the PEI-Sepharose FF resins of low PEI densities. For the PEI-Sepharose FF resins of high ICs (520, 740 and 1220 mmol/L), both protein capacity and De values increased first and then decreased with increasing IS. The increasing trend of protein capacity in the low IS range was considered due to the increase of accessible pores for BSA. The rise–fall trend of De was attributed to the dependencies of the “chain delivery” effect on protein capacity and binding strength, both of which are related to IS. Moreover, the IS sensitivity of the De for the resins of ICs > cIC (740 and 1220 mmol/L) was much higher than those of ICs < cIC, further proving that the “chain delivery” effect in PEI layer did contribute significantly to the overall mass transfer at IC > cIC. Furthermore, the two PEI-Sepharose FF resins of ICs > cIC kept high adsorption capacities and De values up to 200–300 mmol/L NaCl. Therefore, the operating IS ranges for these two PEI-Sepharose FF resins can be much broader than the traditional ion-exchange media.
Co-reporter:Chuan Wang, Yan Sun
Journal of Chromatography A 2013 Volume 1307() pp:73-79
Publication Date(Web):13 September 2013
DOI:10.1016/j.chroma.2013.07.066
•Double-modification strategy was proposed to prepare ion exchangers.•Double-modified composite cryogels possess large surface area and extended tentacles.•Dynamic binding capacity of bovine serum albumin was 1.5–4.6 times enhanced.Composite cryogel monoliths based on poly(2-hydroxyethylmethacrylate) (pHEMA) were fabricated by incorporating polymeric resin particles. The monoliths were sequentially modified by polyethylenimine (PEI) and diethylaminoethyl (DEAE). The novel composite material had rough pore walls and extended anion-exchange tentacles, which provided more binding sites for protein molecules. The dynamic adsorption capacity of bovine serum albumin (BSA) on the novel cryogel bed reached 11.2 mg/mL bed volume at a flow velocity of 8 cm/min, which was about 1.5–4.6 times higher than the cryogel beds obtained by single modifications. The capacity value was also much higher than the BSA capacities of cryogel beds reported in literature (1–6 mg/mL). The capacity decreased only slightly with increasing flow rate from 0.6 to 12 cm/min. The height equivalent to a theoretical plate of the composite beds was in the range 2–2.5 mm, changed indistinctively in a flow rate range 0.6–18 cm/min. Hence, the work has proved that the double-modification strategy was promising for enhancing protein adsorption capacity of cryogel monolith for high-speed protein chromatography.
Co-reporter:Chun-Yan Yang, Ming Li, Xiao-Yan Dong, Yan Sun
Journal of Chromatography A 2013 Volume 1299() pp:85-93
Publication Date(Web):19 July 2013
DOI:10.1016/j.chroma.2013.05.059
•A double modification strategy was developed to enhance charge density of beads.•The double modification enhanced BSA adsorption density of the beads.•Double-modified beads enhanced like-charged protein refolding more significantly.•Refolding yield was independent of the ligand structure.•Beads of higher charge density showed lower salt sensitivity in protein refolding.We have previously found that addition of like-charge media in a refolding system can greatly enhance protein refolding yield, and the media of higher charge density (q) are favorable for facilitating protein refolding. Herein, a double-modification strategy was developed to increase the charge density of mono-sized poly(glycidyl methacrylate) (PGMA) microspheres (0.8 μm). The PGMA beads were firstly modified with poly(ethylenimine) of molecular weight 60,000 (PEIL) or 1200 (PEIS) and then further modified with 2-diethylaminoethylchloride (DEAE) or PEIS. The charge density and bovine serum albumin (BSA) adsorption density (QBSA) of the beads were significantly increased by the double modification. The QBSA of the double-modified beads with the highest q (824 μmol/g) reached 36.3 mg/g, over 35% higher than that of the single-modified beads (26.7 mg/g) with the highest q (300 μmol/g) that a single modification could achieve. The double-modified beads with different ligand structures and charge densities were used for facilitating the refolding of like-charged lysozyme. Lysozyme refolding yield with the single-modified beads of the highest q (300 μmol/g) was 60% at a critical bead concentration (cc) of 100 mg/mL. By contrast, the refolding yields with the double-modified beads of q > 500 μmol/g were 70%, and the refolding yields with the double-modified beads of q ≥ 650 μmol/g could even reach 70% at a cc of 40 mg/mL. This indicates that the double-modified beads of high q values could enhance like-charged protein refolding more significantly than the single-modified beads at a low bead utilization. The facilitating effect of the like-charged beads was independent of the ligand structure. The beads of higher q values showed lower salt sensitivity in protein refolding, and were expected beneficial for use in refolding buffers of higher ionic strengths. The sequential modification strategy for enhancing charge density would help develop more efficient media for protein adsorption and protein refolding applications.
Co-reporter:Lin-Ling Yu, Shi-Peng Tao, Xiao-Yan Dong, Yan Sun
Journal of Chromatography A 2013 Volume 1305() pp:76-84
Publication Date(Web):30 August 2013
DOI:10.1016/j.chroma.2013.07.014
•BSA adsorption to PEI-Sepharose of ionic capacities in 100–1220 mmol/L was studied.•Binding capacity increased with ionic capacity till reaching a plateau at 600 mmol/L.•Protein uptake rate kept at a low level at ionic capacity lower than 600 mmol/L.•Both binding capacity and uptake rate increased drastically at 600–740 mmol/L.•A critical ionic capacity at 600 mmol/L was found.To explore the details of protein uptake to polymer-grafted ion exchangers, Sepharose FF was modified with poly(ethylenimine) (PEI) to prepare anion exchanger of 10 different ionic capacities (ICs, 100–1220 mmol/L). Adsorption equilibria and kinetics of bovine serum albumin (BSA) were then studied. It is found that ionic capacity, i.e., the coupling density of PEI, had significant effect on both adsorption capacity (qm) and effective protein diffusivity (De). With increasing ionic capacity, the qm value increased rapidly at IC < 260 mmol/L and then increased slowly till reaching a plateau at IC = 600 mmol/L. In the IC range of 100–600 mmol/L, however, the De values kept at a low level (De/D0 < 0.07); it first decreased from 0.05 ± 0.01 at IC = 100 mmol/L to 0.01 ± 0.01 at IC = 260 mmol/L and then increased to 0.06 ± 0.01 at IC = 600 mmol/L. Thereafter, sharp increases of the qm and De values [36% (from 201 to 273 mg/mL) and 670% (from 0.06 ± 0.01 to 0.49 ± 0.04), respectively] were observed in the narrow range of IC from 600 to 740 mmol/L. Finally, at IC > 740 mmol/L, the qm value decreased significantly while the De value increased moderately with increasing the IC. The results indicate that PEI chains played an important role in protein adsorption and transport. In brief, there was a critical IC (cIC) or PEI chain density, above which protein adsorption and transport behaviors changed drastically. The cIC was identified to be about 600 mmol/L. Estimation of PEI grafting-layer thickness suggests that PEI chains formed an extended three-dimensional grafting-layer at IC > cIC, which provided high flexibility as well as accessibility of the chains for protein binding. Therefore, at IC > cIC, the adjacent PEI chains became close and flexible enough, leading to facilitated transport of adsorbed protein molecules by the interactions of neighboring chains mediated by the bound molecules. It is regarded as “chain delivery” effect. At the same time, improved accessibility of binding sites led the significant increase of binding capacity. The decrease of qm value at IC > 740 mmol/L is considered due to the decrease of effective porosity. The research has thus provided new insight into protein adsorption and transport in polymer-grafted ion-exchange media.
Co-reporter:Chuan Wang, Xiao-Yan Dong, Zhongyi Jiang, Yan Sun
Journal of Chromatography A 2013 Volume 1272() pp:20-25
Publication Date(Web):11 January 2013
DOI:10.1016/j.chroma.2012.11.059
Novel composite cryogel monoliths were fabricated by incorporating polymeric resin particles and grafting anion-exchange groups on the pore wall surfaces. The embedded resin particles in different size distributions were prepared by grinding poly(glycidyl methacrylate–ethylene glycol dimethacrylate) monoliths. Observations by scanning electron microscopy clearly indicated that the composite cryogel had interconnected large pores (10–100 μm in width), similar to normal (pure) cryogel. However, the composite material had very rough pore walls, which provided larger surface area for protein adsorption. As a result, the dynamic binding capacity of bovine serum albumin (BSA) on the composite cryogel bed reached 6 mg/mL bed (flow velocity, 5 cm/min), which was 2.8 times higher than a cryogel bed without embedding the ground resin particles. The capacity value was also much higher than the BSA capacities of cryogel beds reported in literature (1–4 mg/mL). Though the capacity decreased by about 1 mg/mL with the increase of flow rate from 0.5 to 5 cm/min, it then kept almost unchanged till a flow rate up to 15 cm/min. The height equivalent to a theoretical plate of the composite bed was in the range of 1.1–1.4 mm, and kept nearly constant in a flow rate range of 5–20 cm/min. The results indicated that the composite cryogel bed offered a large improvement in protein adsorption capacity and was suitable for high-speed protein chromatography.Highlights► Composite cryogel monoliths were fabricated by incorporating polymeric resin particles. ► The composite cryogels had rough pore walls that provided larger surface area. ► The dynamic capacity of bovine serum albumin was 2.8 times enhanced. ► The column efficiency kept nearly constant in the flow rate range of 0.5–20 cm/min.
Co-reporter:Qing-Hong Shi;Guo-Dong Jia;Liang Xu
Journal of Separation Science 2013 Volume 36( Issue 18) pp:3075-3085
Publication Date(Web):
DOI:10.1002/jssc.201300297
In this study, a novel column design with a round cross-section was proposed to be suitable for a transverse electric field (EF). Additionally, two beads for entropic interaction chromatography (EIC) were prepared by grafting glycidyl methacrylate onto Toyopearl HW-65F (T65F) beads. Solute partitioning was then investigated to elucidate the role of graft polymerization with and without an EF. In a T65F column, solute partitioning was attributed to the distinct pore structure in the beads and was governed by pore flow. Under EF, partition coefficients (Kp) for solutes decreased with increasing EF strength. In the two EIC columns, a decrease of Kp was also observed without an EF while the fractionation windows were extended. It was more pronounced in the EIC column with a high grafting density (T65F-H). This was explained by the decrease in the effective pore size of solutes caused by the steric hindrance of polymer chains. Under an EF, the solutes showed different partitioning behaviours in the T65F-H column. With increasing EF strength, Kp for vitamin B12 and myoglobin was decreased. In contrast, Kp for large solutes increased as a result of concentration polarization on the bead surface. Both behaviors were related to the modulation of graft polymerization to residual charge on the matrix and the pore size of the solutes.
Co-reporter:Lin Zhang, Ronghong Tang, Shu Bai, Natalie K. Connors, Linda H. L. Lua, Yap P. Chuan, Anton P. J. Middelberg, and Yan Sun
The Journal of Physical Chemistry B 2013 Volume 117(Issue 18) pp:5411-5421
Publication Date(Web):April 15, 2013
DOI:10.1021/jp311170w
Virus-like particles (VLPs) are highly organized nanoparticles that have great potential in vaccinology, gene therapy, drug delivery, and materials science. However, the application of VLPs is hindered by obstacles in their design and production due to low efficiency of self-assembly. In the present study, all-atom (AA) molecular dynamics (MD) simulations coupled with the molecular mechanics-Poisson–Boltzmann surface area (MM-PBSA) method are utilized to examine the molecular interactions in the capsomere of a murine polyomavirus (MPV) VLP. It is found that both low ionic strength and the intracapsomere disulfide bonds are favorable for maintaining a stable capsomere. Simulation results examining the effects of solution conditions on the stabilization of a capsomere were verified by calorimetry experiments. Simulation results of free energy decomposition indicate that hydrophobic interaction is favorable for the formation of a capsomere, whereas electrostatic interaction is unfavorable. With increasing ionic strength, the dominant interaction for the stabilization of a capsomere changes from hydrophobic to electrostatic. By comprehensive analyses, the key amino acid residues (hot spots) in VP1 protein aiding formation of a capsomere in different solution conditions have been identified. These results provide molecular insights into the stabilization of building blocks for VLP and are expected to have implications in their partitioning between the correct and off-pathway reactions in VLP assembly.
Co-reporter:Shuo Wang, Neng Xiong, Xiao-Yan Dong, Yan Sun
Journal of Chromatography A 2013 1320() pp: 118-124
Publication Date(Web):
DOI:10.1016/j.chroma.2013.10.074
Co-reporter:Chun-Yan Yang, Xiao-Yan Dong, Yan Sun
Reactive and Functional Polymers 2013 73(11) pp: 1405-1411
Publication Date(Web):November 2013
DOI:10.1016/j.reactfunctpolym.2013.07.005
Co-reporter:Lin Zhang
Frontiers of Chemical Science and Engineering 2013 Volume 7( Issue 4) pp:456-463
Publication Date(Web):2013 December
DOI:10.1007/s11705-013-1357-y
Hydrophobic charge induction chromatography (HCIC) is a mixed-mode chromatography which is advantageous for high adsorption capacity and facile elution. The effect of the ligand chain length on protein behavior in HCIC was studied. A coarse-grain adsorbent pore model established in an earlier work was modified to construct adsorbents with different chain lengths, including one with shorter ligands (CL2) and one with longer ligands (CL4). The adsorption, desorption, and conformational transition of the proteins with CL2 and CL4 were examined using molecular dynamics simulations. The ligand chain length has a significant effect on both the probability and the irreversibility of the adsorption/desorption. Longer ligands reduced the energy barrier of adsorption, leading to stronger and more irreversible adsorption, as well as a little more unfolding of the protein. The simulation results elucidated the effect of the ligand chain length, which is beneficial for the rational design of adsorbents and parameter optimization for highperformance HCIC.
Co-reporter:Lin-Ling Yu, Yan Sun
Journal of Chromatography A 2012 Volume 1253() pp:105-109
Publication Date(Web):31 August 2012
DOI:10.1016/j.chroma.2012.07.004
Agarose-based matrices have been widely used in ion exchange chromatography (IEC). We have herein observed that positively charged proteins (lysozyme and cytochrome c) are adsorbed on the agarose-based anion-exchangers (Q and DEAE Sepharose FF gels) in a capacity of 10–40 μg/mL. In contrast, negatively charged protein (bovine serum albumin) is not adsorbed to Sepharose FF and SP Sepharose FF gels. Elemental analysis of the gel indicated that the residual anionic sulfate groups in agarose would have worked as the cation exchange groups for the positively charged proteins. The trace adsorption behavior of lysozyme onto Sepharose FF and Sepharose FF-based anion exchangers was studied and the effects of NaCl concentration and cation group density on the adsorption were examined for better understanding of the trace adsorption in chromatographic processes. At NaCl concentrations less than 0.05 mol/L, which is the normal adsorption condition in IEC, the trace adsorption kept at a high level, so this trace adsorption cannot be avoided in the ionic strength range of routine IEC operations. Grafting poly(ethylenimine) (PEI) chain of 60 kDa to a cation group density of 700 mmol/L could reduce the adsorption capacity to about 20 μg/mL, but further reduction was not possible by increasing the cation group density to 1200 mmol/L. Therefore, attentions need to be paid to the phenomenon in protein purification practice using agarose-based matrices. The research is expected to call attentions to the trace adsorption on agarose-based matrices and to the importance in the selection of the suitable solid matrices in the production of high-purity protein products in large-scale bioprocesses.Highlights► Positively charged proteins were adsorbed on Q and DEAE Sepharose FF at 10–40 μg/mL. ► The residual sulfate monoesters on agarose would have caused the trace adsorption. ► Lysozyme adsorption was reduced by grafting poly(ethylenimine) to 700 mmol/L. ► The trace adsorption would compromise product purity and/or recovery.
Co-reporter:Qing-Hong Shi, Zhi-Cong Shi, Yan Sun
Journal of Chromatography A 2012 Volume 1257() pp:48-57
Publication Date(Web):28 September 2012
DOI:10.1016/j.chroma.2012.08.003
Confocal laser scanning microscopy (CLSM) was introduced to visualize particle-scale binary component protein displacement behavior in Q Sepharose HP column. To this end, displacement chromatography of two intrinsic fluorescent proteins, enhanced green fluorescent protein (eGFP) and red fluorescent protein (RFP), were developed using sodium saccharin (NaSac) as a displacer. The results indicated that RFP as well as eGFP could be effectively displaced in the single-component experiments by 50 mmol/L NaSac at 120 and 140 mmol/L NaCl whereas a fully developed displacement train with eGFP and RFP was only observed at 120 mmol/L NaCl in binary component displacement. At 140 mmol/L NaCl, there was a serious overlapping of the zones of the two proteins, indicating the importance of induced-salt effect on the formation of an isotachic displacement train. CLSM provided particle-scale evidence that induced-salt effect occurred likewise in the interior of an adsorbent and was synchronous to the introduction of the displacer. CLSM results at 140 mmol/L NaCl also demonstrated that both the proteins had the same fading rate at 50 mmol/L NaSac in the initial stage, suggesting the same displacement ability of NaSac to both the proteins. In the final stage, the fading rate of RFP in the adsorbent became slow, particularly at lower displacer concentrations. In the binary component displacement, the two proteins exhibited distinct fading rates as compared to the single component displacement and the remarkable lagging of the fading rate was observed in protein displacements. It suggested that the co-adsorbed proteins had significant influence on the formation of an isotachic train and the displacement chromatography of the proteins. Therefore, this research provided particle-scale insight into the dynamic behavior and complexity in the displacement of proteins.Highlights► CLSM was introduced to visualize particle-scale displacement of binary proteins. ► Two native fluorescent proteins were used to establish a novel displacement system. ► Induced-salt effect was synchronous to the application of displacer in particles. ► The research exhibited good relevance between chromatography and CLSM observations.
Co-reporter:Lin-Ling Yu, Xiao-Yan Dong, Yan Sun
Journal of Chromatography A 2012 Volume 1225() pp:168-173
Publication Date(Web):17 February 2012
DOI:10.1016/j.chroma.2011.12.078
We have previously found that addition of charged particles in a refolding solution can greatly increase the refolding yield of like-charged proteins. Herein, porous anion exchangers of different charged group densities, ligand chemistries, pore sizes and particle sizes were prepared with Sepharose FF gel for studying their effects on the oxidative refolding of like-charged lysozyme. We found that charge density had significant contribution to the enhancing effects on lysozyme refolding. At low resin concentration range (<0.04–0.1 g/mL), the refolding yield increased with increasing charged group density and resin concentration. The yield then reached a plateau at a critical resin concentration; the higher the charge density, the lower the critical resin concentration. This implies that gel particles of higher charge density were favorable to offer higher refolding yield at lower added concentrations. In the gel concentration range in which refolding yield has reached plateau, there existed an optimum charge density that gave the highest refolding yield. It was attributed to the electrostatic repulsion effect of the charged groups on the like-charged protein, which reduced the accessible pore volume for the protein. At the same charge density, the refolding yield was independent of ligand chemistry, but a polyelectrolyte group of higher molecular weight was more suitable for grafting the gel to prepare matrices of high charge density. The resins of smaller size exhibited better facilitating effect, and the microporous resin was better than that with superpores. The research is expected to help design more effective charged materials for facilitating protein refolding.Highlights► Refolding yield increased with increasing charged group density and resin concentration. ► The yield then reached a plateau at a critical resin concentration, which decreased with increasing charged group density. ► There existed an optimum charged group density that gave the highest refolding yield. ► High-molecular-weight polyelectrolyte was favorable to prepare high charged group density gels. ► Refolding yield was independent of ligand chemistry.
Co-reporter:Juan Liang;Georg Fieg;Qing-Hong Shi
Journal of Separation Science 2012 Volume 35( Issue 17) pp:2162-2173
Publication Date(Web):
DOI:10.1002/jssc.201200101
Simultaneous and sequential adsorption equilibria of single and binary adsorption of bovine serum albumin and bovine hemoglobin on Q Sepharose FF were investigated in different buffer constituents and initial conditions. The results in simultaneous adsorption showed that both proteins underwent competitive adsorption onto the adsorbent following greatly by protein–surface interaction. Preferentially adsorbed albumin complied with the universal rule of ion-exchange adsorption whereas buffer had no marked influence on hemoglobin adsorption. Moreover, an increase in initial ratios of proteins was benefit to a growth of adsorption density. In sequential adsorption, hemoglobin had the same adsorption densities as single-component adsorption. It was attributed to the displacement of preadsorbed albumin and multiple layer adsorption of hemoglobin. Three isothermal models (i.e. extended Langmuir, steric mass-action, and statistical thermodynamic (ST) models) were introduced to describe the ion-exchange adsorption of albumin and hemoglobin mixtures. The results suggested that extended Langmuir model gave the lowest deviation in describing preferential adsorption of albumin at a given salt concentration while steric mass-action model could very well describe the salt effect in albumin adsorption. For weaker adsorbed hemoglobin, ST model was the preferred choice. In concert with breakthrough data, the research further revealed the complexity in ion-exchange adsorption of proteins.
Co-reporter:Bo Huang, Fu-Feng Liu, Xiao-Yan Dong, and Yan Sun
The Journal of Physical Chemistry B 2012 Volume 116(Issue 1) pp:424-433
Publication Date(Web):December 4, 2011
DOI:10.1021/jp205770p
Protein A from the bacterium Staphylococcus aureus (SpA) has been widely used as an affinity ligand for purification of immunoglobulin G (IgG). The affinity between SpA and IgG is affected differently by salt and pH, but their molecular mechanisms still remain unclear. In this work, molecular dynamics simulations and molecular mechanics Poisson–Boltzmann surface area analysis were performed to investigate the salt (NaCl) and pH effects on the affinity between SpA and human IgG1 (hIgG1). It is found that salt and pH affect the interactions of the hot spots of SpA by different mechanisms. In the salt solution, the compensations between helices I and II of SpA as well as between the nonpolar and electrostatic energies make the binding free energy independent of salt concentration. At pH 3.0, the unfavorable electrostatic interactions increase greatly and become the driving force for dissociation of the SpA–hIgG1 complex. They mainly come from the strong electrostatic repulsions between positively charged residues (H137, R146, and K154) of SpA and the positively charged residues of hIgG1. It is considered to be the molecular basis for hIgG1 elution from SpA-based affinity adsorbents at pH 3.0. The dissociation mechanism is then used to refine the binding model of SpA to hIgG1. The model is expected to help design high-affinity peptide ligands of IgG.
Co-reporter:Shi-Hui Wang, Xiao-Yan Dong, and Yan Sun
The Journal of Physical Chemistry B 2012 Volume 116(Issue 20) pp:5803-5809
Publication Date(Web):April 26, 2012
DOI:10.1021/jp209406t
(−)-Epigallocatechin-3-gallate (EGCG) has been proven effective in preventing the aggregation of amyloid β-protein 42 (Aβ42), and the thermodynamic interactions between Aβ42 and EGCG have been studied in our previous work ( J. Phys. Chem. B 2010, 114, 11576). Herein, to further probe the interactions between different regions of Aβ42 and EGCG, three Aβ42 fragments (i.e., Aβ1–16, Aβ1–30, and Aβ31–42) were synthesized, and the thermodynamic interactions between each of the fragments and EGCG at different EGCG and salt concentrations were investigated by isothermal titration calorimetry. The results indicate that, although hydrogen bonding and hydrophobic interaction are both involved in the interactions between Aβ42 and EGCG, hydrogen bonding mainly happens in Aβ1–16 while hydrophobic interaction mainly happens in Aβ17–42. It is found that when Aβ42 and its fragments are saturated by EGCG, their thermodynamic parameters have linear relationships. The saturated binding stoichiometry (Ns) for Aβ42 is the sum of the Ns values for Aβ1–30 and Aβ31–42, while ΔHs, ΔSs, and ΔGs for Aβ42 are half the sum of the values for Aβ1–30 and Aβ31–42. The result suggests that there are no specific interactions and binding sites in the Aβ42 and EGCG binding. The orders of ΔHs and TΔSs values for the Aβ fragments are determined as Aβ17–42 > Aβ31–42 > Aβ1–30 > Aβ1–16. Moreover, there is significant enthalpy–entropy compensation in the binding of EGCG to Aβ42 and its fragments, resulting in insignificant change of ΔG with the change of the solution environment. The research has shed new light on the molecular mechanisms of the interactions between EGCG and Aβ42.
Co-reporter:Na Zhang, Fu-Feng Liu, Xiao-Yan Dong, and Yan Sun
The Journal of Physical Chemistry B 2012 Volume 116(Issue 24) pp:7040-7047
Publication Date(Web):May 18, 2012
DOI:10.1021/jp300171h
Considerable experimental evidence indicates that trehalose can counteract the denaturing effects of urea on proteins. However, its molecular mechanism remains unknown due to the limitations of current experimental techniques. Herein, molecular dynamics simulations were performed to investigate the counteracting effects of trehalose against urea-induced denaturation of chymotrypsin inhibitor 2. The simulations indicate that the protein unfolds in 8 mol/L urea, but at the same condition the protein retains its native structure in the ternary solution of 8 mol/L urea and 1 mol/L trehalose. It is confirmed that the preferential exclusion of trehalose from the protein surface is the origin of its counteracting effects. It is found that trehalose binds urea via hydrogen bonds, so urea molecules are also expelled from the protein surface along with the preferential exclusion of trehalose. The exclusion of urea from the protein surface leads to the alleviation of the Lennard-Jones interactions between urea and the hydrophobic side chains of the protein in the ternary solution. In contrast, the electrostatic interactions between urea and the protein change little in the presence of trehalose because the decrease in the electrostatic interactions between urea and the protein backbone is canceled by the increase in the electrostatic interactions between urea and the charged side chains of the protein. The results have provided molecular explanations for the counteraction of urea-induced protein denaturation by trehalose.
Co-reporter:Chun-Yan Yang, Lin-Ling Yu, Xiao-Yan Dong, Yan Sun
Reactive and Functional Polymers 2012 72(11) pp: 889-896
Publication Date(Web):November 2012
DOI:10.1016/j.reactfunctpolym.2012.07.016
Co-reporter:Jian Li, Lin Zhang, Yan Sun
Journal of Molecular Graphics and Modelling 2012 Volume 37() pp:49-58
Publication Date(Web):July 2012
DOI:10.1016/j.jmgm.2012.04.002
Molecular interactions between the von Willebrand factor (VWF) A1 domain and glycoprotein Ibα (GPIbα) promote the initial adhesion of platelets and subsequent arterial thrombus formation. However, little is understood about the interactions at a molecular level. Therefore, the binding dynamics and involved molecular interactions between VWF A1 domain and GPIbα in both water and physiological saline are investigated using molecular dynamics simulations and all-atom models. Faster binding is observed in water than that in physiological saline, and patches of opposite charges are observed at the binding interface. Moreover, molecular mechanics-Poisson–Boltzmann surface area analysis indicates that the binding is promoted by the long-range electrostatic interactions and then maintained by hydrophobic interactions. For the initial binding, the hot spots include the residues E14, E128, D175, D83, E151, D106, D63, E5, D18, E225, D235 in GPIbα, and K608, K569, K644, R571, K572, R636, K599 in VWF A1 domain. For the final complex formation, however, 72% of the favorable contributions are from hydrophobic interactions. The results provided molecular insight into the initial platelet adhesion. The hot spots identified would be beneficial for developing novel drugs for thrombotic diseases.Graphical abstract.Highlights► Molecular interactions between VWF A1 domain and GPIbα are investigated. ► Binding is promoted by the long-range electrostatic interactions. ► Final complex formation is maintained by the hydrophobic interactions. ► The hot spots for the initial binding are all charged residues. ► Favorable hydrophobic contribution from M239 is significant for complex formation.
Co-reporter:Guofeng Zhao, Lin Zhang, Shu Bai, Yan Sun
Separation and Purification Technology 2011 Volume 82() pp:138-147
Publication Date(Web):27 October 2011
DOI:10.1016/j.seppur.2011.09.002
Displacement chromatography is a separation technique with high capacity and high resolving power. Mass transfer kinetics is an essential factor that determines the separation efficiency of displacement chromatography, but few studies have been reported on the kinetics of the displacement process, especially at the particle level. In this work, the kinetics of protein and displacer adsorptions, protein displacement and adsorbent regeneration in hydrophobic charge induction displacement chromatography were studied by both stirred-batch experiments and online visualization with confocal laser scanning microscopy (CLSM). Stirred-batch experiments showed that small-molecule displacers had higher effective diffusivities and mass transfer rates than proteins. For the displacement systems used in this work, the rate of displacement was dependent on the mass transfer and adsorption rate of the displacer. Direct visualization of the displacement process was achieved by labeling the protein with fluorescein isothiocyanate (FITC) and by using a fluorescent displacer, rhodamine 6G. The concentration profiles obtained by CLSM clearly revealed the higher intraparticle mass transfer rate of the displacer and subsequent high rate of displacement. The observations by CLSM were mostly consistent with the results of the stirred-batch processes. However, although the direct visualizations by CLSM has provided valuable information of the microscopic processes in hydrophobic charge induction displacement chromatography, efforts are still needed to develop an improved displacement model system (protein and displacer) that can provide more accurate and quantitative information of displacement processes.Graphical abstractHighlights► Microscopic process of HCIDC was examined by multiple techniques. ► Consistent results were observed in stirred-batch experiment and CLSM observation. ► Valuable information has been provided for the inherent mechanisms of HCIDC.
Co-reporter:Qing-Hong Shi, Guo-Dong Jia, Yan Sun
Separation and Purification Technology 2011 Volume 77(Issue 3) pp:375-381
Publication Date(Web):4 March 2011
DOI:10.1016/j.seppur.2011.01.006
We have integrated immobilized metal affinity chromatography (IMAC) with preparative electrochromatography to study the intensification effects of electric field (EF) on the IMAC process for protein binding and purification. A seven-compartment chromatographic column with longitudinal (L), oscillatory transverse (T) or two-dimensional (2D) electric fields was designed for the studies. IMAC experiments were conducted to assess the performance of the electrochromatography and to optimize protein adsorption conditions. Compared with the dynamic binding capacity (DBC) in normal mode without an EF (nEF), the DBC for bovine serum albumin increased by 23%, 67% and 130% in modes of L-, T- and 2D-EF, respectively. This confirmed that EF had great contribution to the intensification of IMAC process. Such phenomenon of intensification caused by EF was also of universality and unrelated to metal ions immobilized on the adsorbents. In the purification of alcohol dehydrogenase (ADH) from clarified yeast extract, the loading volume in the mode of 2D-EF could be increased to twice that in the mode of nEF. At the double feed loading condition, the application of 2D-EF still led to significant improvement in the purification performance, such as the increase of activity yield from 65.4% to 84.6%, specific activity from 15.9 U/mg to 17.5 U/mg, and purification factor from 6.36 to 7.04. Therefore, the application of 2D-EF could strikingly intensify the IMAC performance and the IMAC in 2D-EF mode exhibited a good potential in protein purification from complex feedstocks.Graphical abstractResearch highlights▶ Immobilized metal affinity chromatography was integrated with two-dimensionalelectric fields (2D-EF) for the intensification of protein purification process. ▶ The dynamic binding capacity of protein could be 2.3 times increased with the 2D-EF. ▶ In the purification of alcohol dehydrogenase, the loading volume in the mode of 2D-EF could be twice increased. ▶ The application of 2D-EF led to significant improvement in the purification performance at the double feed loading condition.
Co-reporter:Bo Huang, Fu-Feng Liu, Xiao-Yan Dong, and Yan Sun
The Journal of Physical Chemistry B 2011 Volume 115(Issue 14) pp:4168-4176
Publication Date(Web):March 22, 2011
DOI:10.1021/jp111216g
Protein A (SpA) affinity chromatography has been widely used for the purification of immunoglobulin G (IgG). However, the molecular mechanism of the affinity between IgG and SpA remains unclear. In this work, molecular dynamics simulations and molecular mechanics—Poisson−Boltzmann surface area analysis were performed to investigate the molecular mechanism of the affinity interactions. It is found that hydrophobic interaction contributes more than 80% to the binding free energy, while electrostatic interaction plays a minor role (<20%). Through free energy decomposition and pair interaction analysis, the hot spots of the SpA−hIgG1 complex are identified. For hIgG1, the hot spots include the residues of I253, H310, Q311, D315, K317, E430, and N434. For SpA, residues F132, Y133, H137, E143, R146, and K154 contribute significantly. Furthermore, helix I of SpA binds Fc through hydrophobic interaction, while helix II mainly provides electrostatic interaction that determines the binding selectivity to different Igs. Finally, the binding motif of SpA is constructed, which would help design novel high-affinity ligands of IgG.
Co-reporter:Fu-Feng Liu, Xiao-Yan Dong, Lizhong He, Anton P. J. Middelberg, and Yan Sun
The Journal of Physical Chemistry B 2011 Volume 115(Issue 41) pp:11879-11887
Publication Date(Web):September 8, 2011
DOI:10.1021/jp202640b
Considerable experimental evidence indicates that (−)-epigallocatechin-3-gallate (EGCG) inhibits the fibrillogenesis of Aβ42 and alleviates its associated cytotoxicity. However, the molecular mechanism of the inhibition effect of EGCG on the conformational transition of Aβ42 remains unclear due to the limitations of current experimental techniques. In this work, molecular dynamics simulations and molecular mechanics-Poisson–Boltzmann surface area (MM-PBSA) analysis were coupled to better understand the issue. It was found that the direct interactions between EGCG and the peptide are the origin of its inhibition effects. Specifically, EGCG molecules expel water from the surface of the Aβ42, cluster with each other, and interact directly with the peptide. The results of free energy decomposition calculated by MM-PBSA indicate that the nonpolar term contributes more than 71% to the binding free energy of the EGCG-Aβ42 complex, while polar interactions (i.e., hydrogen bonding) play a minor role. It was identified that there are 12 important residues of Aβ42 that strongly interact with EGCG (Phe4, Arg5, Phe19, Phe20, Glu22, Lys28, Gly29, Leu34-Gly37, and Ile41), while nonpolar interactions are mainly provided by the side chains of some hydrophobic residues (Phe, Met and Ile) and the main chains of some nonhydrophobic residues (Lys28 and Gly29). On the contrary, polar interactions are mainly formed by the main chain of Aβ42, of which the main chains of Gly29 and Gly37 contribute greatly. The work has thus elucidated the molecular mechanism of the inhibition effect of EGCG on the conformational transition of Aβ42, and the findings are considered critical for exploring more effective agents for the inhibition of Aβ42 fibrillogenesis.
Co-reporter:Lin Zhang, Shu Bai, Yan Sun
Journal of Molecular Graphics and Modelling 2011 Volume 29(Issue 7) pp:906-914
Publication Date(Web):June 2011
DOI:10.1016/j.jmgm.2011.02.004
Modeling, especially the force field, is crucial for the accuracy of molecular dynamics (MD) simulations. In order for more accurate description of adsorption and desorption behaviors of lysozyme in hydrophobic charge induction chromatography (HCIC), the Martini coarse-grained (CG) force field has been modified based on the statistical analysis and comparison of an all-atom (AA) force field, GROMOS96 43A1, and the Martini force field. The parameters describing the protein–adsorbent interactions have been adjusted to avoid too strong and unrealistic adsorption of lysozyme on the agarose matrix and HCIC ligands. It is found that the adsorption and desorption behaviors monitored using the modified Martini force field and MD simulation are consistent with previous simulation results with 46-bead β-barrel model protein. Repeated adjustment of both protein position and orientation is necessary to generate enough contacts for a stable adsorption. After reducing the pH in the mobile phase, the lysozyme–ligand electrostatic repulsion leads to protein desorption. In the adsorption process, little conformational transition of lysozyme is observed due to its stable structure, which is in line with previous experimental observations. So, it is concluded that after appropriate modification, the Martini force field can be used to examine the HCIC process of lysozyme. The modification strategy has thus extended the applicability of the Martini force field to protein chromatography, and it is expected to facilitate studies of exploring the molecular details in adsorption chromatography of proteins.Graphical abstractHighlights► Statistical analysis provides suitable modification of Martini force field. ► The modified Martini force field gives correct description of lysozyme in HCIC. ► Lysozyme has little conformational transition within adsorbent pore. ► Modification extends the applicability of Martini to protein chromatography.
Co-reporter:Kai-Feng Du, Shu Bai, Xiao-Yan Dong, Yan Sun
Journal of Chromatography A 2010 Volume 1217(Issue 37) pp:5808-5816
Publication Date(Web):10 September 2010
DOI:10.1016/j.chroma.2010.07.046
Agarose gels were fabricated by water-in-oil emulsification with the addition of CaCO3 granules at 8–16 wt%. Thus agarose beads of different superporosities were produced after dissolving the solid porogen. The superporous agarose (SA) and homogeneous agarose gels were double cross-linked and modified with diethylaminoethyl chloride to produce anion exchangers. We have proposed to use a superporous replica (porous titania microspheres) to examine the superporous structure and pore size distribution of the soft gel. The replica was prepared with the agarose gel entrapping CaCO3 granules by a sol–gel-templating method. It was found that the superpores created by CaCO3 granules were uniformly distributed and ranged from 0.95 μm to 1.33 μm. The physical properties of the gels were significantly affected by the porogen content. Importantly, by increasing the solid porogen to 12 wt%, the bed permeability and effective porosity increased about 48% and 33%, respectively. Further increase in the porogen to 16 wt% led to a decrease of the mechanical strength. With increasing superpores in the beads, the dynamic adsorption capacity of the packed columns increased obviously at 305–916 cm/h. Besides, the column efficiency changed less with increasing flow velocity up to 1200 cm/h. It was concluded that the use of 12 wt% CaCO3 granules in agarose solution was beneficial for the fabrication of the SA gel with good mechanical stability and promising performance for protein chromatography.
Co-reporter:Qing-Hong Shi, Guo-Dong Jia, Yan Sun
Journal of Chromatography A 2010 Volume 1217(Issue 31) pp:5084-5091
Publication Date(Web):30 July 2010
DOI:10.1016/j.chroma.2010.05.065
A novel chromatographic medium for high-capacity protein adsorption was fabricated by grafting dextran (40 kDa) onto the pore surfaces of superporous agarose (SA) beads. The bead was denoted as D-SA. D-SA, SA and homogeneous agarose (HA) beads were modified with sulfopropyl (SP) group to prepare cation exchangers, and the adsorption and uptake of lysozyme on all three cation-exchange chromatographic beads (SP-HA, SP-SA and SP-D-SA) were investigated at salt concentrations of 6–50 mmol/L. Static adsorption experiments showed that the adsorption capacity of SP-D-SA (2.24 mmol/g) was 78% higher than that of SP-SA (1.26 mmol/g) and 54% higher than that of SP-HA (1.45 mmol/g) at a salt concentration of 6 mmol/L. Moreover, salt concentration had less influence on the adsorption capacity and dissociation constant of SP-D-SA than it did on SP-HA, suggesting that dextran-grafted superporous bead is a more potent architecture for chromatographic beads. In the dynamic uptake of lysozyme to the three cation-exchange beads, the De/D0 (the ratio of effective pore diffusivity to free solution diffusivity) values of 1.6–2.0 were obtained in SA-D-SA, indicating that effective pore diffusivities of SP-D-SA were about two times higher than free solution diffusivity for lysozyme. At 6 mmol/L NaCl, the De value in SA-D-SA (22.0 × 10−11 m2/s) was 14.4-fold greater than that in SP-HA. Due to the superior uptake kinetics in SA-D-SA, the highest dynamic binding capacity (DBC) and adsorption efficiency (the ratio of DBC to static adsorption capacity) was likewise found in SP-D-SA. It is thus confirmed that SP-D-SA has combined the advantages of superporous matrix structure and drafted ligand chemistry in mass transport and offers a new opportunity for the development of high-performance protein chromatography.
Co-reporter:Kai-Feng Du, Xiao-Yan Dong, and Yan Sun
Industrial & Engineering Chemistry Research 2010 Volume 49(Issue 24) pp:12560-12566
Publication Date(Web):November 16, 2010
DOI:10.1021/ie901727s
Agarose gel entrapping CaCO3 granules is used for the synthesis of bimodal porous CaTiO3 beads by a method that combines a sol−gel process and solid phase reaction, in which CaCO3 (1.05 μm) performed as both calcium resource and porogen for creating macropores. The amount of CaCO3 granules embedded in agarose gel is varied from 0 to 16 wt %, and the macropores on the surface and in the inner of final CaTiO3 beads are investigated. It is found that the addition of 12 wt % of CaCO3 produced CaTiO3 beads that present interconnected macropores of about 745 ± 20 nm. The calcination temperature affects the surface area, mesopore size, phase state, and crystalline size of the CaTiO3 beads. By calcination at 450 °C, the CaTiO3 beads prepared by the addition of 12 wt % CaCO3 are amorphous with an average pore size of 9 nm and possess a high surface area of about 225 m2 g−1. With an increase of temperature to 600, 750, and 900 °C, the CaTiO3 is transformed into perovskite in size of about 32, 34, and 37 nm with mesopores of about 20, 23, and 24 nm, respectively, and its specific surface area decreases below 20 m2 g−1. The bimodal porous CaTiO3 beads of about 15 μm are packed into a column for investigating its chromatographic performance. The column shows much higher column efficiency than that packed with CaTiO3 beads without removal of the porogen. The remarkably high separation performance is attributed to the unique surface property and interconnected macropores present in the CaTiO3 beads.
Co-reporter:Shi-Hui Wang, Fu-Feng Liu, Xiao-Yan Dong and Yan Sun
The Journal of Physical Chemistry B 2010 Volume 114(Issue 35) pp:11576-11583
Publication Date(Web):August 18, 2010
DOI:10.1021/jp1001435
One of the key factors of Alzheimer’s disease (AD) is the conversion of amyloid β-peptide (Aβ) from its soluble random coil form into various aggregated forms. (−)-Epigallocatechin-3-gallate (EGCG) has been proved effective in preventing the aggregation of Aβ, but the thermodynamic mechanisms are still unclear. In this work, isothermal titration calorimetry (ITC) was utilized to study the interactions between Aβ42 and EGCG at different temperatures, salt concentrations, pH values, and EGCG and Aβ42 concentrations. Molecular dynamics (MD) simulations were performed to study the hydrogen bonding between Aβ42 and EGCG. The results indicate that the binding stoichiometry N is linearly related to the EGCG/Aβ42 ratio. Hydrophobic interaction and hydrogen bonding are both substantial in the binding process, but the extent of their contributions changes with experimental conditions. Namely, the predominant interaction gradually shifts from a hydrogen bonding to a hydrophobic interaction with the increase of the EGCG/Aβ42 ratio, resulting in a transition of the binding from enthalpy-driven to entropy-driven. This experimental observation is validated by the MD simulations. The binding of EGCG to Aβ42 can be promoted by increasing temperature and salt concentration and changing pH away from Aβ42′s pI. The findings have provided new insight into the molecular interactions between Aβ42 and EGCG from a thermodynamic perspective and are expected to facilitate the research on the inhibition of Aβ42 aggregation.
Co-reporter:Lin Zhang, Shu Bai, Yan Sun
Journal of Molecular Graphics and Modelling 2010 Volume 28(Issue 8) pp:863-869
Publication Date(Web):June 2010
DOI:10.1016/j.jmgm.2010.03.006
Hydrophobic charge induction chromatography (HCIC) is an adsorption chromatography combining hydrophobic interaction in adsorption with electrostatic repulsion in elution. Ligand density has significant effects on protein adsorption behavior, but little is understood about the effect of ligand homogeneity on surface morphology of ligands, protein conformational transition and dynamics within adsorbent pore due to the lack of microscopic experimental techniques. In the present study, a coarse-grained adsorbent pore model established in an earlier work is used to represent the actual porous adsorbent composed of matrix and immobilized HCIC ligands. Two adsorbent pores with different ligand distributions are constructed by adjusting the coupling sites, denoted as L1 and L2. In L1 the ligands are bonded uniformly while in L2 the ligands are arranged in lines in the axial direction and thus exhibit a heterogeneous distribution. Protein adsorption, desorption, and conformational transition in both L1 and L2 are shown by molecular dynamics simulations of a 46-bead β-barrel coarse-grained model protein within the adsorbent pore models. The simulations indicate that ligand homogeneity has significant effect on both the irreversibility and the dynamics of adsorption while no obvious effect on protein conformation distribution. In comparison with L1, L2 leads to irreversible and slow adsorption, indicating the strict requirement of a suitable protein orientation to reach stable adsorption. The simulations have provided new insight into the microscopic behavior of HCIC, which would be beneficial to the rational design of adsorbents and parameter optimization for high-performance HCIC.
Co-reporter:Lin Zhang, Guofeng Zhao and Yan Sun
The Journal of Physical Chemistry B 2010 Volume 114(Issue 6) pp:2203-2211
Publication Date(Web):January 25, 2010
DOI:10.1021/jp903852c
High ligand density is usually required in hydrophobic charge induction chromatography (HCIC) for high adsorption capacity. However, it is not clear to what extent the ligand density alters the adsorption and desorption behaviors, or if this leads to the protein conformational transition within adsorbent pores. In the present study, molecular dynamics simulation is performed to examine the effects of ligand density in HCIC using a 46-bead β-barrel coarse-grained model protein and a coarse-grained adsorbent pore model established in our earlier work. Four ligand densities (1.474, 1.769, 2.212, and 2.949 μmol/m2) are simulated at 298.15 K. The simulations indicate that both the capacity and irreversibility of adsorption increase with ligand density. However, it is found that the fastest adsorption occurs at a ligand density of 2.212 μmol/m2 rather than at the highest density studied. Analyses of adsorption trajectories, protein−ligand interaction energy, and the free energy map indicate that there is repulsion of protein when unfavorable contacts of the protein and ligands occur. There is an enhanced repulsion at 2.949 μmol/m2, which increases the energy barrier to the transition region and reduces the opportunities to get stable adsorption, thus leading to the decreased adsorption rate. At 2.212 μmol/m2, however, the repulsion is mild and the high ligand coverage provides abundant opportunities for the protein to get the fastest adsorption and thus causes the maximum unfolding. In the following simulations, complete and irreversible desorption is observed at all ligand densities, in agreement with the easy pH-induced elution behavior of HCIC observed experimentally. It is found that there is a suitable balance between hydrophobic attraction and electrostatic repulsion at 2.212 μmol/m2, which leads to the slowest desorption kinetics and causes the maximum unfolding. Moreover, analysis of unfolded protein distribution indicates that unfolding occurs mainly on the ligand surface in both adsorption and desorption. The behaviors have been comprehensively elucidated by molecular and thermodynamic analyses.
Co-reporter:Xiao-Yan Dong, Li-Jun Chen, Yan Sun
Journal of Chromatography A 2009 Volume 1216(Issue 27) pp:5207-5213
Publication Date(Web):3 July 2009
DOI:10.1016/j.chroma.2009.05.008
This article has proposed an artificial chaperone-assisted immobilized metal affinity chromatography (AC-IMAC) for on-column refolding and purification of histidine-tagged proteins. Hexahistidine-tagged enhanced green fluorescent protein (EGFP) was overexpressed in Escherichia coli, and refolded and purified from urea-solubilized inclusion bodies by the strategy. The artificial chaperone system was composed of cetyltrimethylammonium bromide (CTAB) and β-cyclodextrin (β-CD). In the refolding process, denatured protein was mixed with CTAB to form a protein–CTAB complex. The mixture was then loaded to IMAC column and the complex was bound via metal chelating to the histidine tag. This was followed by washing with a refolding buffer containing β-CD that removed CTAB from the bound protein and initiated on-column refolding. The effect of the washing time (i.e., on-column refolding time) on mass and fluorescence recoveries was examined. Extensive studies by comparison with other related refolding techniques have proved the advantages of AC-IMAC. In the on-column refolding, the artificial chaperone system suppressed protein interactions and facilitated protein folding to its native structure. So, the on-column refolding by AC-IMAC led to 99% pure EGFP with a fluorescence recovery of 80%. By comparison at a similar final EGFP concentration (0.6–0.8 mg/mL), this fluorescence recovery value was not only much higher than direct dilution (14%) and AC-assisted refolding (26%) in bulk solutions, but also superior to its partner, IMAC (60%). The operating conditions would be further optimized to improve the refolding efficiency.
Co-reporter:Qing-Hong Shi, Zheng Cheng, Yan Sun
Journal of Chromatography A 2009 Volume 1216(Issue 33) pp:6081-6087
Publication Date(Web):14 August 2009
DOI:10.1016/j.chroma.2009.06.065
4-(1H-imidazol-1-yl) aniline (AN) was immobilized on Sepharose CL-6B (AN-Sepharose) for use as a new ligand of mixed-mode chromatography. Adsorption equilibria of immunoglobulin G (IgG) and bovine serum albumin (BSA) to AN-Sepharose were studied at extensive pH values (4.0–8.8) and salt concentrations (0–1.0 mol/L). Static binding studies indicated that AN-Sepharose had a good salt-tolerance property for IgG adsorption up to 1.0 mol/L NaCl. This was attributed to the combined ligand–protein interactions (hydrophobic interaction, hydrogen bonding and charge transfer interaction). By contrast with BSA, AN-Sepharose showed a high binding selectivity for IgG at NaCl > 0.2 mol/L. Dynamic binding capacities (DBC) of IgG and BSA at 10% breakthrough were measured at pH 4.0–8.8 by frontal analysis chromatography. IgG had DBC values over 40 mg/mL at pH 7.0–8.8, and the maximum reached 59 mg/mL at pH 8.0. At pH 5.0, a distinct drop in DBC to 8.5 mg/mL was observed, but that for BSA kept over 22 mg/mL. The result suggested that IgG could be selectively desorbed from AN-Sepharose by decreasing pH to about 5. Therefore, compared to BSA, AN-Sepharose exhibited a dual-selectivity for IgG in both adsorption and elution. Purification of IgG from bovine serum also confirmed the dual-selectivity. IgG purity of the pooled fractions by elution at pH 4.0, 4.5 and 5.0 reached 55% and the highest purity, 80%, was obtained at pH 4.5. The average purification factor of IgG was over 25. The results indicate that AN is a promising ligand of mixed-mode chromatography for antibody purification from a complex feedstock.
Co-reporter:Liang Xu, Xiao-Yan Dong, Yan Sun
Journal of Chromatography A 2009 Volume 1216(Issue 32) pp:6071-6076
Publication Date(Web):7 August 2009
DOI:10.1016/j.chroma.2009.06.032
A new method for protein analysis, that is, electroosmotic pump-assisted capillary electrophoresis (EOPACE), is developed and demonstrated to possess several advantages over other CE-based techniques. The column employed in EOPACE consists of two linked sections, poly(vinyl alcohol) (PVA)-coated and uncoated capillaries. The PVA-coated capillary column is the section for protein electrophoresis in EOPACE. Electroosmotic flow (EOF) is almost completely suppressed in this hydrophilic polymer coated section, so protein electrophoresis in the PVA-modified capillary is free of irreversible protein adsorption to the capillary inner wall. The uncoated capillary section serves as an electroosmotic pump, since EOF towards cathode occurs at neutral pH in the naked silica capillary. By the separation of a protein mixture containing cytochrome c (Cyt-c), myoglobin and trypsin inhibitor, we have demonstrated the advantages of EOPACE method over other relevant ones such as pressure assisted CE, capillary zone electrophoresis (CZE) with naked capillary and CZE with PVA-coated capillary. A significant feature of EOPACE is that simultaneous separation of cationic, anionic and uncharged proteins at neutral pH can be readily accomplished by a single run, which is impossible or difficult to realize by the other CE-based methods. The high column efficiency and good reproducibility in protein analysis by EOPACE are verified and discussed. In addition, separation of tryptic digests of Cyt-c with the EOPACE system is demonstrated.
Co-reporter:Wei Yuan, Yan Sun
Separation and Purification Technology 2009 Volume 68(Issue 1) pp:109-113
Publication Date(Web):30 June 2009
DOI:10.1016/j.seppur.2009.04.002
Four anion-exchangers of different ion-exchange capacities (21–129 μmol/mL) were prepared by coupling diethylaminoethyl to Sepharose 6FF. The adsorbents were used to study the dynamic protein adsorption in ion-exchange electrochromatography with an oscillatory transverse electric field perpendicular to the mobile-phase flow (IEEC). The static adsorption capacity of bovine serum albumin (BSA) increased from 67 to 186 mg/mL in the ionic capacity range, but the effective pore diffusion coefficient of the protein decreased from 10.9 × 10−12 to 1.85 × 10−12 m2/s with increasing the ionic capacity due to the hindrance effect of the bound protein molecules at the pore entrance. So, the dynamic binding capacity (DBC) of protein in ion-exchange chromatography decreased with increasing the ionic capacity. By applying an electric field of 30 mA, the DBC in IEEC packed with ion-exchangers of high ionic capacities (53 and 129 μmol/mL) increased significantly (over 30% and 100%, respectively). This was because the high surface charge density led to high electroosmotic flow that enhanced intraparticle mass transfer in IEEC. In comparison, the DBC in IEEC packed with ion-exchangers of low ionic capacities (21 and 35 μmol/mL) increased only slightly (ca. 10%) under the same condition. The results indicated the minor effect of electrophoretic mobility on the intraparticle mass transfer. Hence, it is beneficial to use ion-exchangers of high ionic capacity for high-capacity purification of proteins by IEEC.
Co-reporter:Kai-Feng Du, Dong Yang and Yan Sun
Industrial & Engineering Chemistry Research 2009 Volume 48(Issue 2) pp:755
Publication Date(Web):December 3, 2008
DOI:10.1021/ie8011165
Porous titania beads were synthesized by a sol−gel templating method using agarose gel as template. The morphology and structure of the materials were characterized by X-ray diffraction, scanning electron microscopy, high-resolution transmission electron microscopy, and N2 adsorption analysis, etc. It was found that the size and pore structure of the resultant titania beads could be readily controlled by using various sized templates and by changing the precipitation conditions. Under optimized conditions (i.e., impregnating agarose gel into titanium precursors for 6 h, three cycles of repeated impregnation and hydrolyzing process, and calcination at 450 °C), porous titania beads of 7−150 μm in mean diameters were fabricated by using different sized agarose beads as templates. The beads displayed perfect spherical shape, with an average pore size of 6 nm and specific surface area of 69.9 m2 g−1. The titania beads of 15 μm in average diameter were further characterized for application as liquid chromatographic packings. Flow hydrodynamic experiment indicated that they possessed high mechanical strength to withstand a pressure drop up to 12 MPa. The column efficiency reached 6165 plates m−1 for acetone and 4650 plates m−1 for N,N-dimethylaniline under nonretained condition. Moreover, baseline separation of three aniline derivatives featuring only small structural differences was realized by using this packed column.
Co-reporter:Lin Zhang, Guofeng Zhao and Yan Sun
The Journal of Physical Chemistry B 2009 Volume 113(Issue 19) pp:6873-6880
Publication Date(Web):April 17, 2009
DOI:10.1021/jp809754k
Hydrophobic charge induction chromatography (HCIC) is an adsorption chromatography combining hydrophobic interaction in adsorption with electrostatic repulsion in elution. The method has been successfully applied in the separation and purification of antibodies and other proteins. However, little is understood about protein conformational transition and the dynamic process within adsorbent pores. In the present study, a pore model is established to represent the realistic porous adsorbent composed of matrix and immobilized HCIC ligands. Protein adsorption, desorption, and conformational transition in the HCIC pore and its implications to the separation performance are shown by a molecular dynamics simulation of a 46-bead β-barrel coarse-grained model protein in the adsorbent pore. Repeated adjustment of both protein position and orientation is observed before reaching a stable adsorption. Once the protein is adsorbed, there is a dynamic equilibrium between unfolding and refolding. The effect of hydrophobic interaction strength between protein and ligands on adsorption phenomena is then examined. Strong hydrophobic interaction, representing the presence of high-concentration lyotropic salt in mobile phase, can speed up the adsorption but cause protein unfolding more significantly. On the contrary, weak hydrophobic interaction, representing the absence of a lyotropic salt or the presence of a chaotropic agent, can reserve native protein conformation but does not lead to stable adsorption. In the elution, protein unfolding occurs due to simultaneous hydrophobic adsorption and electrostatic repulsion in the opposite directions. When the protein has been desorbed, the conformational transition between unfolded and native protein is still observed due to the long-range nature of electrostatic interaction. The simulation has provided molecular insight into protein conformational transition in the whole HCIC process, and it would be beneficial to the rational design of ligands and parameter optimizations for high-performance HCIC.
Co-reporter:Fu-Feng Liu, Luo Ji, Xiao-Yan Dong and Yan Sun
The Journal of Physical Chemistry B 2009 Volume 113(Issue 32) pp:11320-11329
Publication Date(Web):July 21, 2009
DOI:10.1021/jp905580j
Soluble amyloid oligomers are a cytotoxic species in Alzheimer’s disease, and the recent discovery that trehalose can prohibit aggregation of amyloid β-peptide (Aβ) has received great attention. However, its inhibition mechanism remains unclear. In order to investigate the molecular mechanism of the inhibition effect, molecular dynamics simulations of Aβ16−22 and Aβ40 peptides at different trehalose concentrations (0−0.18 mol/L) are performed using an all-atom model. The simulations confirmed that Aβ16−22 aggregation is prevented by trehalose in a dose-dependent manner, and it is found that the preferential exclusion effect of trehalose is the origin of its inhibition effects. Namely, there is preferential hydration on the peptide surface (3 Å), and trehalose molecules cluster around the peptides at a distance of 4−5 Å. At high trehalose concentrations, the preferential exclusion of trehalose leads to three sequential effects that prevent the nucleation and elongation of Aβ16−22 oligomers. First, the secondary structures of Aβ16−22 monomers are stabilized in the turn, bend, or coil, so the β-sheet-rich structure that is prone to forming peptide oligomers is prevented. Second, the thin hydration layer and trehalose clusters can weaken hydrophobic interactions that lead to Aβ16−22 aggregation. Third, more direct and indirect H-bonds form between trehalose and Aβ16−22, which suppress the interpeptide hydrogen bonding. Analyses of the simulation data for a single Aβ40 peptide indicate that trehalose can inhibit the nucleation and elongation of Aβ40 by a similar mechanism with that on Aβ16−22 oligomerization. The work has thus elucidated the molecular mechanism of trehalose on the inhibition of Aβ oligomeric aggregation.
Co-reporter:Liang Xu, Xiao-Yan Dong, Yan Sun
Journal of Chromatography A 2009 1216(48) pp: 8567
Publication Date(Web):
DOI:10.1016/j.chroma.2009.09.067
Co-reporter:Dongmei Wang;Guodong Jia;Liang Xu
Frontiers of Chemical Science and Engineering 2009 Volume 3( Issue 3) pp:229-234
Publication Date(Web):2009 September
DOI:10.1007/s11705-009-0213-6
Anion-exchange superporous cellulose (DEAE-SC) and microporous cellulose (DEAE-MC) adsorbents were packed in an electrochromatographic column, and the effect of external electric field (eEF) on the dynamic adsorption was investigated. The column was designed to provide longitudinal, transverse or 2-dimensional (2D) eEF. It was found that the electro-kinetic effect caused by the introduction of an electric field played an important role in the dynamic adsorption of bovine serum albumin to the adsorbents. The dynamic binding capacity (DBC) in the presence of 2D eEF was higher than in the presence of a one-dimensional eEF. The effect of flow velocity on the DBC of the two adsorbents was also demonstrated. It was found that the effect of electric field on the DEAE-MC column was more remarkable than that on the DEAE-SC column at the same flow rate, whereas the DEAE-SC column showed higher DBC and adsorption efficiency (AE) than the DEAE-MC column. With increasing flow rate, the DEAE-SC column could still offer high DBC and AE in the presence of the 2D eEF. For example, a DBC of 21.4 mg/mL and an AE of 57.7% were obtained even at a flow rate as high as 900 cm/h. The results indicate that the 2D electrochromatography packed with the superporous cellulose adsorbent is promising for high-speed protein chromatography.
Co-reporter:Hai-Ping Zhang, Shu Bai, Liang Xu, Yan Sun
Journal of Chromatography B 2009 Volume 877(Issue 3) pp:127-133
Publication Date(Web):15 January 2009
DOI:10.1016/j.jchromb.2008.11.026
Mono-sized magnetic polyglycidyl methacrylate (PGMA) microspheres cross-linked by divinylbenzene were fabricated by the dispersion polymerization and penetration–deposition method. The magnetic beads were nonporous, spherical, and mono-sized (1.0 μm). Moreover, it was superparamagnetic with a saturation magnetization of 16.5 emu/g. The prepared magnetic beads were then functionalized with high-density amino groups and used as anion exchangers to capture plasmid DNA from concentrated bacterial lysates of Escherichia coli without treatment with ribonuclease A. The adsorption capacity of plasmid DNA reached 90 μg/mg resin in the buffer solution of pH 6.5 and 0.5 mol/l NaCl content. The product was of high recovery yield (91.7% DNA) and of high purity with almost 100% protein and 62.5% RNA removal. Recycled use of the magnetic material for plasmid purification exhibited stability of the magnetic microspheres. The magnetic beads were also effective in the purification of DNA from original crude bacterial lysates. For example, 19.4 μg plasmid DNA (A260/A280 = 1.90) was isolated from 1.5 ml cell culture. The results presented in this article suggest that the prepared mono-sized magnetic adsorbent is suitable for high efficient isolation and purification of plasmid DNA from crude feedstock with high recovery yield and purity.
Co-reporter:Dong-Hao Zhang, Shu Bai, Meng-Yuan Ren, Yan Sun
Food Chemistry 2008 Volume 109(Issue 1) pp:72-80
Publication Date(Web):1 July 2008
DOI:10.1016/j.foodchem.2007.12.020
Response surface methodology was successfully applied to optimize lipase-catalyzed enantioselective esterification of (±)-menthol. The effects of various reaction conditions, including reaction time, temperature, enzyme loading, substrate molar ratio and water activity, were investigated. A Central Composite Rotatable Design was employed to search for the optimal conversion of (±)-menthol and enantiomeric excess. A quadratic polynomial regression model was used to analyze the experimental data at a 95% confidence level (p < 0.05). The analysis confirmed that reaction temperature, enzyme loading and reaction time were the significant factors affecting the conversion of (±)-menthol. Moreover, reaction temperature, enzyme loading, substrate molar ratio and reaction time were found to affect the enantiomeric excess significantly. The coefficient of determination of these two models was found to be 0.980 and 0.967, respectively. Two sets of optimum reaction conditions were established and the verified experimental trials were performed for validating the optimum points. Under the optimum conditions, the conversion of (±)-menthol and the enantiomeric ratio exceeded 53% and 40%, respectively.
Co-reporter:Guo-Dong Jia, Xiao-Yan Dong, Yan Sun
Separation and Purification Technology 2008 Volume 59(Issue 3) pp:277-285
Publication Date(Web):1 March 2008
DOI:10.1016/j.seppur.2007.06.019
A novel preparative electrochromatographic column with seven compartments was developed. The design gave promise to provide transverse, longitudinal or two-dimensional (2D) electric field on the column. The central gel compartment of 2 mL was packed with dye–ligand affinity adsorbent. Bovine serum albumin (BSA) was used as a model protein to investigate the dynamic adsorption behavior in the electrochromatography. The effects of various operating variables indicated that the electroosmotic flow on the charged adsorbent surface could promote the intraparticle mass transport of the protein, leading to the increase of its dynamic binding capacity (DBC). Moreover, it was confirmed that the 2D electric field promoted the mass transport more efficiently than a 1D electric filed. Hence, the DBC of BSA was 4.5 times increased with the 2D electric filed as compared to the 2.4 times increase in a 1D electric field. The results have proven that the 2D electrochromatography is promising for high capacity protein adsorption.
Co-reporter:Meng-Yuan Ren, Shu Bai, Dong-Hao Zhang and Yan Sun
Journal of Agricultural and Food Chemistry 2008 Volume 56(Issue 7) pp:2388-2391
Publication Date(Web):March 14, 2008
DOI:10.1021/jf073067f
Magnetic DEAE-GMA-EDMA microspheres were prepared via suspension polymerization and used for the immobilization of Candida rugosa lipase by ion exchange. The effect of pH values on the immobilization of lipase was investigated. Resolution of (±)-menthol in the hydrophobic ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate was performed by immobilized lipase-catalyzed enantioselective esterification with propionic anhydride as acyl donor. The effects of pH condition at lipase immobilization on the conversion and enantioselectivity were investigated. As a result, pH memory of the immobilized lipase for catalyzing (±)-menthol resolution in the ionic liquid was observed. Better conversion and the best enantioselectivity were obtained with the immobilized lipase prepared at pH 5.0. Under the condition, (−)-menthyl propionate with enantiomeric excess of >90% was obtained. Moreover, the enantioselectivity of the immobilized lipase decreased gradually with increasing pH value.
Co-reporter:Yan Sun, Kun Yang
Separation and Purification Technology 2008 Volume 60(Issue 2) pp:180-189
Publication Date(Web):20 April 2008
DOI:10.1016/j.seppur.2007.08.004
Parallel mass transport of protein in the pore fluid and on the pore wall of porous adsorbent is modeled based on the Maxwell–Stefan theory that uses chemical potential gradient as the diffusive driving force. The uptake kinetics of proteins (BSA and γ-globulin) to anion exchanger is studied by batch adsorption. The parallel diffusion model based on the MS approach (MS-ParD model) and its simplified form, the surface diffusion model (MS-SD model), are analyzed and compared with those based on the Fick's law (F-ParD and F-SD models). It is found that the models from the MS equation and the Fick's law are quite different from each other. For the MS-ParD model, both the pore and surface diffusion coefficients are constant, while the surface or pore diffusion coefficient for the F-ParD model varies significantly with initial protein concentration. Moreover, the MS-SD model can be used to describe the uptake of a protein that shows nearly a rectangular isotherm, in which the surface diffusion contributes very small to the mass transport. In addition, an equation that describes the relationship between the surface diffusion coefficients for the MS-SD and F-SD models is proposed. It is observed that the ratio of the surface diffusion coefficients for the MS-ParD and F-ParD models for less favorably adsorbed protein can be qualitatively described by a theoretical equation. It is concluded that the MS-ParD model is superior to the F-ParD model for describing the non-ideality of adsorbed protein.
Co-reporter:Fu-Feng Liu, Xiao-Yan Dong, Yan Sun
Journal of Molecular Graphics and Modelling 2008 Volume 27(Issue 4) pp:421-429
Publication Date(Web):November 2008
DOI:10.1016/j.jmgm.2008.07.002
Recent work has shown that trehalose can facilitate and inhibit protein folding, but little is known about the molecular basis of these effects. Molecular-level insights into how the osmolyte affects protein folding are of significance for the rational design of small molecular additives for enhancing or hindering the folding of proteins. To investigate the molecular mechanisms of the facilitation and inhibition effects of trehalose on protein folding, molecular dynamics (MD) simulation of a β-hairpin peptide (Trp-Arg-Tyr-Tyr-Glu-Ser-Ser-Leu-Glu-Pro-Glu-Pro-Asp) in different trehalose concentrations (0–0.26 mol/L) is performed using an all-atom model. It is found that at a proper trehalose concentration (0.065 mol/L), the peptide folds faster than that in water, but it cannot fold to the β-hairpin at higher trehalose concentrations. Free energy landscape analysis indicates the presence of three intermediate states in both pure water and in 0.065 mol/L trehalose, but the potential energy barriers in the folding pathway decrease greatly in 0.065 mol/L trehalose, so the peptide folding is facilitated. Moreover, at this trehalose concentration, there is a favorable balance between the peptide backbone hydrogen bonds (H-bonds) and the peptide-trehalose H-bonds, leading to the stabilization of the folded peptide. At higher trehalose concentrations, however, trehalose molecules cluster in the peptide region and interact with the peptide via many H-bonds that prevent the peptide from folding to its native structure. The energy landscape analysis indicates that the potential energy barriers increase so greatly that the peptide cannot overcome it, getting trapped in a local free energy basin. The work reported herein has elucidated the molecular mechanism of the peptide folding in the presence of trehalose.
Co-reporter:Zheng Guo, Yan Sun
Food Chemistry 2007 Volume 100(Issue 3) pp:1076-1084
Publication Date(Web):2007
DOI:10.1016/j.foodchem.2005.11.011
Enzymatic production of a homogeneous 1,3-diglyceride of polyunsaturated fatty acids (PUFAs) was carried out using Novozym 435 as biocatalyst and conjugated linoleic acid (CLA) as a model fatty acid. Three different operation modes, namely, magnetic stirring under vacuum, vacuum-driven N2 bubbling and incubation with molecular sieves, were examined to find an efficient protocol for the enzymatic production. Studies on the effects of mass transfer showed that the occurrence of mass transfer limitation was strongly dependent on the operational modes. Vacuum-driven N2 bubbling proved to be capable of eliminating mass transfer resistance, creating effective interaction for a multiple-phase reaction system and yielding an efficient water removal and a faster reaction rate. Hence, vacuum-driven N2 stirring was considered as the best choice among the tested strategies for the production of pure 1,3-diglyceride of PUFAs with industrial interests, because it gave a higher yield of the desired product, higher productivity and lower impurity content due to its suppression of the acylmigration of 1,3-diglyceride to 1,2-diglyceride. The yield of 92–96% 1,3-dCLG could be obtained when 5 mmol of glycerol were incubated with 10–12 mmol CLA for about 3 h at 45–55 °C and a pressure less than 10 mbar, with enzyme loading of 40–70 g l−1. Among the operational parameters, temperature and reaction time were found to have profound effects on the acylmigration and yield of 1,3-diglyceride. Moreover, the enzyme showed excellent operational stability in this protocol under the optimized conditions (little activity loss of enzyme was observed after 10 consecutive batch reactions), indicating the potential of this technology for industrial application.
Co-reporter:Yang Liu, Xiao-Yan Dong, Yan Sun
Separation and Purification Technology 2007 Volume 53(Issue 3) pp:289-295
Publication Date(Web):1 March 2007
DOI:10.1016/j.seppur.2006.08.001
The reversed micellar solution formed by Sorbitan trioleate (Span 85) coupled with Cibacron Blue F3G-A (CB) as an affinity ligand show good potential for use in reversed micellar protein extraction operations. In this work, the extraction behavior of lysozyme and ovalbumin with the reversed micellar phase has been investigated at extensive pH values and CB concentrations. The partitioning isotherms of lysozyme and ovalbumin were expressed by the Langmuir equation. By addition of 3 vol.% hexanol to the reversed micellar phase, the extraction capacity of lysozyme reached 6.38 mg/mL, over seven times higher than that of ovalbumin (0.83 mg/mL). Thus, lysozyme was separated from crude chicken ovalbumin solutions with a purification factor over 20. Moreover, the reversed micellar solution was recycled three times for the separation, and the purification factor was kept nearly 20 in the recycling. Incomplete back extraction of lysozyme to the stripping solution compromised the total recovery yields in the following recycles, so more effective back extraction method needs to be developed to make the extraction system more attractive. In general, the present results have shown the potential of the affinity-based reversed micellar system for application in protein separations.
Co-reporter:Guo-Yong Sun;Zheng Yang;Xiao-Yan Dong
Journal of Applied Polymer Science 2007 Volume 103(Issue 1) pp:17-23
Publication Date(Web):23 OCT 2006
DOI:10.1002/app.23872
Rigid biporous beads (BiPB) were fabricated by double emulsification. An aqueous suspension of superfine calcium carbonate granules and organic solvent were used as porogenic agents to create superpores and micropores, respectively. The polymerization of monomers, glycidyl methacrylate, and ethylene glycol dimethacrylate was initiated with benzoin ethyl ether by ultraviolet irradiation. Modified with diethylamine (DEA), the BiPB were derivatized into an anion-exchange medium (which is denoted as DEA–BiPB). The DEA–BiPB with an average diameter of 46.3 μm was characterized to possess two types of pores, that is, micropores (20–200 nm) and superpores (500–5300 nm). Flow hydrodynamic experiments showed that the DEA–BiPB column had a smaller backpressure than that of the conventional microporous beads column at a given flow rate. The static adsorption capacity of the DEA–BiPB was close to that of the DEA–MiPB for bovine serum albumin. However, frontal analysis demonstrated that the dynamic binding capacity of the DEA–BiPB column was two times higher than that of the DEA–MiPB at a flow rate of 1800 cm/h. Moreover, the purification of the molecular chaperone GroEL was carried out with the DEA–BiPB column at two flow rates (150 and 1500 cm/h). This showed that the GroEL purification was nearly the same at the two flow rates tested. These results indicate that the DEA–BiPB column is promising for high-speed protein chromatography. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 103: 17–23, 2007
Co-reporter:Yuan Li;Xiao-Yan Dong;Yuan Li;Xiao-Yan Dong
Journal of Applied Polymer Science 2007 Volume 104(Issue 4) pp:2205-2211
Publication Date(Web):27 FEB 2007
DOI:10.1002/app.24417
A biporous absorbent coupled with mercaptopyridine was synthesized for the purification of plasmid DNA. Analyses by scanning electron microscopy and mercury intrusion porosimetry revealed that the matrix contained two families of pores, i.e., micropores and superpores. The superpores provided not only convective flow channels for the mobile phase, but also a large surface for biomacromolecules binding. So the chromatographic process can be operated at high flow rate with high column efficiency and low backpressure as identified on a 2-mL column. When 10 mL crude feedstock containing 3 mg of plasmid (5.4 kb pcDNA3) was loaded at a flow rate as high as 20 cm/min, the separation was finished in 10 min, and the plasmid was completely recovered with undetectable impurities of nucleic acids and proteins. The productivity was determined to be 9.0 g/L h, comparable to the pDNA productivity obtained using the commercial column. These results indicate that the biporous medium is promising for high-throughput purification of plasmid DNA. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 104: 2205–2211, 2007
Co-reporter:Xiaopeng Zhou;Xueli Su
Frontiers of Chemical Science and Engineering 2007 Volume 1( Issue 2) pp:103-112
Publication Date(Web):2007 May
DOI:10.1007/s11705-007-0020-x
A study of nonlinear competitive adsorption equilibria of proteins is of fundamental importance in understanding the behavior of preparative chromatographic separation. This work describes the nonlinear binary protein adsorption equilibria on ion exchangers by the statistical thermodynamic (ST) model. The single-component and binary protein adsorption isotherms of bovine hemoglobin (Hb) and bovine serum albumin (BSA) on SP Sepharose FF were determined by batch adsorption experiments in 0.05 mol/L sodium acetate buffer at three pH values (4.5, 5.0 and 5.5) and three NaCl concentrations (0.05, 0.10 and 0.15 mol/L) at pH 5.0. The ST model was found to depict the effects of pH and ionic strength on the single-component equilibria well, with model parameters depending on the pH and ionic strength. Moreover, the ST model gave acceptable fitting to the binary adsorption data with the fitted single-component model parameters, leading to the estimation of the binary ST model parameter. The effects of pH and ionic strength on the model parameters are reasonably interpreted by the electrostatic and thermodynamic theories. Results demonstrate the availability of the ST model for describing nonlinear competitive protein adsorption equilibria in the presence of two proteins.
Co-reporter:Shu Bai, Zheng Guo, Wei Liu, Yan Sun
Food Chemistry 2006 Volume 96(Issue 1) pp:1-7
Publication Date(Web):May 2006
DOI:10.1016/j.foodchem.2005.01.047
Lauric acid-stabilized magnetic particles were prepared by coprecipitation in the presence of lauric acid and used for the covalent immobilization of Candida rugosa lipase via carbodiimide activation. Size analysis by transmission electron microscopy (TEM) and measurement of magnetization curves revealed that the immobilized lipase was superparamagnetic. Resolution of (±)-menthol was performed by the immobilized lipase-catalyzed enantioselective esterification with propionic anhydride. Effects of various reaction parameters, such as enzyme load, solvents, water activity, substrate concentration, reaction time and temperature, on the conversion as well as enantioselectivity were investigated. As a result, (−)-menthyl propionate with a yield higher than 96% and over 88% enantiomeric excess of products was obtained. Better conversion and enantioselectivity could be expected for the immobilized lipase-catalyzed reaction performed at 30 °C for 2.5 h with 0.2 mol/l of (±)-menthol. Hexane was found to be the most suitable solvent, and the activity as well as enantioselectivity of the immobilized lipase decreased gradually with increasing water activity. Good durability of the immobilized lipase to catalyze the resolution of (±)-menthol was also observed.
Co-reporter:Yi Yuan, Shu Bai, Yan Sun
Food Chemistry 2006 Volume 97(Issue 2) pp:324-330
Publication Date(Web):July 2006
DOI:10.1016/j.foodchem.2005.04.020
Enantioselective esterification of (±)-menthol was studied using Candida rugosa lipase (CRL) in ionic liquids (1-butyl-3-methyl-imidazolium hexafluorophosphate ([BMIM][PF6]) and 1-butyl-3-methyl-imidazolium tetraflouroborate) and organic solvents of different hydrophobicities. Propionic anhydride was employed as an acylating agent. Because the enzyme showed comparable conversion yield and enantioselectivity in [BMIM][PF6] and hexane in a 24-h reaction, more work focused on these two reaction media. Comparison of the activity, stability and enantioselectivity of CRL was carried out by examining the effects of the mole ratio of substrates, temperature, incubation time and enzyme recycling. It was found that temperature control was more crucial in the ionic liquid than in hexane to reach high conversion and enantioselectivity. The ionic liquid system showed an advantage of using less acid anhydride to achieve higher (±)-menthol conversion yield and better enantioselectivity. Moreover, during an incubation of 4–60 days in the ionic liquid, CRL activity was 2.5 times higher than its initial value, while that in hexane decreased to less than 60% in 2 days. In addition, the enzyme showed potentiality of recycled use in the ionic liquid. These advantages of the ionic liquid suggest that it would be used as a green alternative to organic solvents for the enantioselective esterification of (±)-menthol.
Co-reporter:Guo-Min Tan;Xiao-Yan Dong
Journal of Separation Science 2006 Volume 29(Issue 5) pp:684-690
Publication Date(Web):14 MAR 2006
DOI:10.1002/jssc.200500354
Protein separations by a novel size-exclusion electrochromatography (SEEC) are presented. The present SEEC, denoted as pSEEC, was established with an oscillatory low-voltage electric field perpendicular to the mobile-phase streamline. Retention experiments with different proteins indicated that the influence of electric field strength on the partition coefficient is different for different proteins as well as for the same protein under different mobile-phase conditions. These results of protein retention led to the experimental design of protein separations with binary mixtures of BSA and immunoglobulin G (IgG), myoglobin (Myo) and lysozyme (Lys), as well as ovalbumin (Oval) and Myo. The separation results for the binary protein systems sufficiently exhibited the applicability of the pSEEC for various separations in terms of their molecular weights (MWs) as well as pIs. For example, it was possible to separate the gel-excluded proteins (BSA/IgG) as well as gel-permeable and similar-molecular-weight proteins (Myo/Lys) by the pSEEC. Moreover, in the cases of Oval/Myo, which could be partially separated by size-exclusion chromatography, the use of the pSEEC greatly improved the resolution and the separation became possible at high sample loading. The results indicate that the pSEEC technology is promising for preparative protein separations.
Co-reporter:Guo-Feng Zhao;Xiao-Yan Dong;Wei Yuan
Journal of Separation Science 2006 Volume 29(Issue 15) pp:2383-2389
Publication Date(Web):6 SEP 2006
DOI:10.1002/jssc.200600149
The ion-exchange electrochromatography with an oscillatory electric field perpendicular to the mobile-phase flow driven by pressure (pIEEC) was used to separate hen egg-white (HEW) proteins. The results were compared with those of normal ion-exchange chromatography (IEC). The column was designed as three-compartment rectangular column of 2-mL with dimensions (length×width×depth) of 40×10×5 mm3 and the electric field was applied across the direction of column width. Q Sepharose FF was packed into the central compartment as the chromatographic bed. It was confirmed that the dynamic binding capacity (DBC) of different proteins (ovotransferrin and ovalbumin) in the HEW solution increased 2.3 times when an oscillatory electric current of 30 mA at 1/20 Hz was applied in the transverse column direction. Then, the HEW proteins were separated by the pIEEC at loading amounts 2.3-fold higher than those by the IEC. When the feedstock of about one-third of the DBC was applied to the columns (i. e., 7 mL for the pIEEC and 3 mL for the IEC), similar separation efficiencies of the two chromatographic modes were achieved. Both the recovery yield and purity reached 73% to over 90%. The results indicate that the pIEEC is promising for high-capacity purification of proteins.
Co-reporter:Guofeng Zhao, Fuqiang Li, Guanying Peng, Qinghong Shi, Yan Sun
Journal of Biotechnology (October 2008) Volume 136(Supplement) pp:
Publication Date(Web):1 October 2008
DOI:10.1016/j.jbiotec.2008.07.054
Co-reporter:Ming Lu, Shu Bai, Kun Yang, Yan Sun
China Particuology (February–April 2007) Volume 5(Issues 1–2) pp:180-185
Publication Date(Web):1 February 2007
DOI:10.1016/j.cpart.2007.01.012
Non-porous magnetic polymer microspheres with a core–shell structure were prepared by a novel micro-suspension polymerization technique. A stable iron oxide ferrofluid was used to supply the magnetic core, and the polymeric shell was made of glycidyl methacrylate (GMA monomer) and ethylene dimethacrylate (cross-linker). In the preparation, polyvinyl alcohol was used as the stabilizer, and a lauryl alcohol mixture as the dispersant. The influence of various conditions such as aqueous phase volume, GMA and initiator amounts, reaction time and stirring speed on the character of the microspheres was investigated. The magnetic microspheres were then characterized briefly. The results indicate that the microspheres with active epoxy groups had a narrow size distribution range from 1 to 10 μm with a volume-weighted mean diameter of 4.5 μm. The saturation magnetization reached 19.9 emu/g with little coercivity and remanence.
Co-reporter:Kun Yang, Shu Bai, Yan Sun
Chemical Engineering Science (August 2008) Volume 63(Issue 16) pp:4045-4054
Publication Date(Web):1 August 2008
DOI:10.1016/j.ces.2008.05.013
A self-contained research system based on the technique of confocal laser scanning microscopy (CLSM) was put up to quantitatively analyze the dynamics of protein adsorption to porous cation exchanger by mathematical modeling. Bovine serum albumin adsorption to the cation exchanger SP Sepharose FF was performed by batch adsorption and micro-flow cell in which protein concentration in single absorbent was visualized by CLSM. The effects of ionic strength and the protein concentration in liquid phase (50 mmol/L acetate buffer, pH 5.0) on the adsorption dynamics were examined. The intraparticle concentration profile data experimentally obtained from CLSM were quantitatively analyzed by three diffusive mass transfer models (i.e., pore diffusion, surface diffusion and Maxwell–Stefan models (MSM)) in virtue of the attenuation equation for the CLSM visualization developed earlier. The nuance between the model simulations and experimental results of the developing protein distribution in a single adsorbent particle could thus be found out. Without salt addition to the buffer, the adsorption isotherm was strongly favorable, and the pore diffusion model (PorDM) and MSM gave similarly good simulations of the experiments, whereas the surface diffusion model was unreasonable in the model presumption. Moreover, it was observed that the experimentally obtained adsorption front was relative flatter as compared with the calculated results from the PorDM, which implied the possible existence of surface diffusion. With increasing salt concentration, the simulations became to deviate from the experiments. Especially, when the salt concentration approached 50 mmol/L, all the three mass transfer models could hardly give good simulation of the experiment. This was considered due to the difference in adsorption behavior between the fluorescence labeled and unlabeled proteins therein.
Co-reporter:Qing-Hong Shi, Guo-Dong Jia, Yan Sun
Separation and Purification Technology (4 March 2011) Volume 77(Issue 3) pp:375-381
Publication Date(Web):4 March 2011
DOI:10.1016/j.seppur.2011.01.006
We have integrated immobilized metal affinity chromatography (IMAC) with preparative electrochromatography to study the intensification effects of electric field (EF) on the IMAC process for protein binding and purification. A seven-compartment chromatographic column with longitudinal (L), oscillatory transverse (T) or two-dimensional (2D) electric fields was designed for the studies. IMAC experiments were conducted to assess the performance of the electrochromatography and to optimize protein adsorption conditions. Compared with the dynamic binding capacity (DBC) in normal mode without an EF (nEF), the DBC for bovine serum albumin increased by 23%, 67% and 130% in modes of L-, T- and 2D-EF, respectively. This confirmed that EF had great contribution to the intensification of IMAC process. Such phenomenon of intensification caused by EF was also of universality and unrelated to metal ions immobilized on the adsorbents. In the purification of alcohol dehydrogenase (ADH) from clarified yeast extract, the loading volume in the mode of 2D-EF could be increased to twice that in the mode of nEF. At the double feed loading condition, the application of 2D-EF still led to significant improvement in the purification performance, such as the increase of activity yield from 65.4% to 84.6%, specific activity from 15.9 U/mg to 17.5 U/mg, and purification factor from 6.36 to 7.04. Therefore, the application of 2D-EF could strikingly intensify the IMAC performance and the IMAC in 2D-EF mode exhibited a good potential in protein purification from complex feedstocks.Graphical abstractDownload full-size imageResearch highlights▶ Immobilized metal affinity chromatography was integrated with two-dimensionalelectric fields (2D-EF) for the intensification of protein purification process. ▶ The dynamic binding capacity of protein could be 2.3 times increased with the 2D-EF. ▶ In the purification of alcohol dehydrogenase, the loading volume in the mode of 2D-EF could be twice increased. ▶ The application of 2D-EF led to significant improvement in the purification performance at the double feed loading condition.
Co-reporter:Xiao-Yan Dong, Jian-Hua Liu, Fu-Feng Liu, Yan Sun
Biochemical Engineering Journal (15 March 2009) Volume 43(Issue 3) pp:
Publication Date(Web):15 March 2009
DOI:10.1016/j.bej.2008.10.015
Osmolyte molecules such as betaine and trehalose are protein stabilizers while l-arginine (Arg) and guanidine hydrochloride (GdnHCl) are the most widely used aggregation suppressor in protein refolding. We have herein studied the effects of the osmolyte molecules and l-arginine together with GdnHCl (0–6 mol/L) on the intermolecular interaction of native and denatured lysozyme by self-interaction chromatography. The self-interaction is characterized in terms of the osmotic second virial coefficient (B) of the protein, the increase of which represents the decrease of intermolecular attraction of the protein. It is found that the effect of Arg on the self-interaction of lysozyme is similar with GdnHCl, but its competence is much weaker than the denaturant. At higher GdnHCl concentrations (>0.5 mol/L), Arg can be used to suppress the self-association of lysozyme. In contrast to Arg, B increases with increasing betaine or trehalose concentration at the GdnHCl concentration range studied. The results indicate the cooperativity of each osmolyte with GdnHCl, and the different mechanisms of their effects from Arg on the B values. The work confirms that the osmolytes are not only protein stabilizers, but also protein aggregation suppressors for both native and denatured protein molecules.
Co-reporter:Lin Zhang, Yan Sun
Biochemical Engineering Journal (15 February 2010) Volume 48(Issue 3) pp:408-415
Publication Date(Web):15 February 2010
DOI:10.1016/j.bej.2009.12.003
Co-reporter:Liang Xu, Xiao-Yan Dong, Yan Sun
Biochemical Engineering Journal (15 December 2010) Volume 53(Issue 1) pp:137-142
Publication Date(Web):15 December 2010
DOI:10.1016/j.bej.2010.10.005
Co-reporter:Xiao-Yan Dong, Li-Jun Chen, Yan Sun
Biochemical Engineering Journal (15 December 2009) Volume 48(Issue 1) pp:65-70
Publication Date(Web):15 December 2009
DOI:10.1016/j.bej.2009.08.007
Co-reporter:Guo-Zhen Wang, Xiao-Yan Dong, Yan Sun
Biochemical Engineering Journal (15 August 2011) Volume 55(Issue 3) pp:169-175
Publication Date(Web):15 August 2011
DOI:10.1016/j.bej.2011.04.002
Oxidative protein refolding is a big challenge for the recovery of disulfide bonds containing recombinant proteins expressed as inclusion bodies. In this study, the disulfide form of a small peptide mimic of protein disulfide isomerase, CGC, was explored to facilitate oxidative protein refolding with residual dithiothreitol (DTT) as the reductant. Working with DTT, CGC gave higher yield and folding rate than oxidized glutathione. Then, a new pentapeptide, RKCGC disulfide, was designed. It was found that RKCGC was more effective than CGC in increasing the folding rate and final yield, as demonstrated in the oxidative refolding of lysozyme and ribonuclease A. It was because the pentapeptide has lower pKa and higher reduction potential than CGC. Moreover, it efficiently facilitated protein refolding not only at neutral pH but also in a weak alkaline buffer. With CGC or RKCGC as the oxidant, the DTT removal step and the addition of other thiol reductants were not needed. This would benefit in decreasing the processing time and cost in protein refolding processes. This design has thus provided more efficient oxidant, and it may pave the way for the design of more effective thiol-disulfide redox agents.Highlights► A new oxidant, RKCGC disulfide, was designed to catalyze oxidative protein refolding. ► RKCGC facilitated oxidative protein refolding more effectively than CGC disulfide. ► RKCGC was effective not only in neutral pH but also in weak alkaline solutions. ► The design reveals that adding nearby positive charges to peptide disulfide is effective to improve its catalytic activity by lowering the pKa of the corresponding thiol.
Co-reporter:Guofeng Zhao, Xiao-Yan Dong, Yan Sun
Journal of Biotechnology (12 October 2009) Volume 144(Issue 1) pp:3-11
Publication Date(Web):12 October 2009
DOI:10.1016/j.jbiotec.2009.04.009
Mixed-mode chromatography is a chromatographic method that utilizes more than one form of interactions between the stationary phase and the solutes in a feed stream. Compared with other types of chromatography, mixed-mode chromatography is advantageous in its salt-independent adsorption, facile elution by charge repulsion, and unique selectivity. Hence, it has already proved beneficial for the separation of proteins as well as other purposes. In this article, mixed-mode ligands for protein purification have been reviewed. These ligands usually have an aliphatic or aromatic group as the hydrophobic moiety and an amino, carboxyl or sulfonic group as the ionic moiety. Heterocyclic groups are good ligand candidates for their unique hydrophobicity and dissociation property. Hydrogen bonding groups also have influences on the performance of mixed-mode adsorbents. These principles should be considered in the screening and design of mixed-mode ligands. Strategies for the design of synthetic affinity ligands, especially the bioinformatics and combinatorial methods, may be adopted for mixed-mode ligand design. More efforts are needed for the development of rational design and screening methods for mixed-mode protein ligands by sophisticated computational and experimental approaches.
Co-reporter:Guo-Zhen Wang, Xiao-Yan Dong, Yan Sun
Biochemical Engineering Journal (1 September 2009) Volume 46(Issue 1) pp:7-11
Publication Date(Web):1 September 2009
DOI:10.1016/j.bej.2009.04.005
Co-reporter:Hongchen Liu, Linling Yu, Xiaoyan Dong, Yan Sun
Journal of Colloid and Interface Science (1 April 2017) Volume 491() pp:
Publication Date(Web):1 April 2017
DOI:10.1016/j.jcis.2016.12.038
Fibrillogenesis of amyloid β-protein (Aβ) in human brain has been implicated as the main cause of Alzheimer’s disease. A few small molecules from natural sources have been discovered for their inhibition effects on Aβ fibrillation, of which (−)-epigallocatechin-3-gallate (EGCG) is one of the most broadly investigated compounds for its potent inhibitory activity. However, in vivo applicability of the inhibitors is largely limited by their low transmembrane efficiency. Hence, it is of great significance to develop inhibition systems that function at low concentrations. In this work, a dual-inhibitor system containing EGCG and negatively charged polymeric nanoparticles (NP10), which was also demonstrated effective on the inhibition of Aβ aggregation, was developed and comprehensively studied by extensive biophysical and biological assays. It was found that the dual-inhibitor system at low concentrations was more effective on the inhibition and detoxification of Aβ (Aβ42 and Aβ40) fibrillation than the additive effects of these two agents working individually. Namely, there was a synergistic effect of the two inhibitors. The synergism factor reached 1.25 with 5 μM EGCG and 5 μg/mL NP10. Kinetic studies with Aβ40 revealed that the two inhibitors functioned in different Aβ assembling stages: NP10 mainly inhibited primary nucleation, while EGCG suppressed fibril elongation and changed the fibril structure to make it show less seeding activities in the secondary nucleation. NP10 might also help EGCG binding to Aβ, leading to its enhanced inhibitory effects on fibril elongation and secondary nucleation. The synergistic effect observed in the dual-inhibitor system offered new insight into the development of potent inhibitor systems against amyloid neurotoxicity.
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