Heparosan, the capsular polysaccharide discovered in many pathogenic bacteria, is a promising material for heparin preparation. In this study, the Pasteurella multocida heparosan synthase 1 (PmHS1) module was used to synthesize heparosan with controlled molecular weight, while tuaD/gtaB module or gcaD module was responsible for UDP-precursors production in Bacillus subtilis 168. After metabolic pathway optimization, the yield of heparosan was as high as 237.6 mg/L in strain containing PmHS1 module and tuaD/gtaB module, which indicated that these two modules were key factors in heparosan production. The molecular weight of heparosan varied from 39 to 53 kDa, which indicated that heparosan molecular weight could be adjusted by the amount of PmHS1 and the ratio of two UDP precursors. The results showed that it would be possible to produce safe heparosan with appropriate molecular weight which is useful in heparin production.

•SERP1 has complicated structure due to its composition of nine monosaccharides.•SERP1 shows high hypoglycemic properties in diabetic mice.•SERP1 could be explored as a functional food or supplement from industrial waste.An acidic polysaccharide (SERP1) was isolated from the residue of Sarcandra glabra (Thunb.) Nakai by water extraction and purified by decoloration and ion exchange chromatography. The structure of SERP1 was determined by HPLC, HPSEC-MALLS, FT-IR, and NMR. The results indicated that SERP1 was a homogeneous heteropolysaccharide with the absolute molecular weight of 4.208 × 104 Da in the aqueous phase. 1,4-linked α-d-galacturonic acid, methyl esterified 1,4-linked α-d-galacturonic acid, 1,4-linked α-d-glucuronic acid, 1,5-linked α-l-arabinose, 1,3-linked β-d-galactose 1,4-linked α-d-glucose, 1,4,6-linked β-d-glucose, 1,6-linked β-d-glucose, and 1,2-linked rhamnose existed in SERP1. In vitro α-glucosidase inhibition assay indicated that SERP1 had a low IC50 value of 49.01 μg/mL, which exhibited stronger α-glucosidase enzyme inhibitory activity than acarbose at the same concentration. The treatment of SERP1 to type 2 diabetes mellitus mice alleviated the hyperglycemia, increased glucose utilization of peripheral tissues of the liver and inhibited the liver injury. This study provides a possible exploration to use valuable industrial waste.

Insufficient sialylation can result in rapid clearance of therapeutic glycoproteins by intracellular degradation, which is mainly mediated by asialoglycoprotein receptors (ASGPRs) on hepatic cells. In contrast, for glycoproteins, a long half-life is often related to high level of terminal sialic acid. These could be extremely important for insufficient sialylated biomedicines in clinic, and development of therapeutic glycoproteins in laboratory. However, how the desialylated glycoproteins are removed and how to evaluate the ASGPRs mediated endocytosis in vitro needs further investigate. Herein we described an integrative characterization of ASGPRs in vitro to elucidate its endocytosis properties. The endocytosis was determined by a fluorescence-based quantization method. The results showed that the ASGPRs could bind to poorly sialylated glycoproteins including asialofetuin and low sialylated recombinant Factor VIIa with a relatively higher ASGPRs binding affinity, and induce a more rapid endocytosis in vitro. Moreover, the mechanism under the internalization of ASGPRs was also investigated, which was found to depend on clathrin and caveolin. Utilizing the relative fluorescence quantification can be suitable for measurement of insufficient sialylated glycoprotein endocytosis and quality control of therapeutic glycoproteins, which could be useful for the understanding of the development of therapeutic glycoproteins.

•A purified acidic polysaccharide was obtained from fruits of Lycium barbarum L.•The structural characteristics were investigated by a combination of chemical and instrumental analysis approaches along with a degradation pattern study.•The fine primary structure and chain conformation of this polysaccharide was elucidated.An acidic polysaccharide, named as p-LBP, was isolated from Lycium barbarum L. by water extraction and purified by decoloration, ion exchange chromatography, dialysis and gel chromatography, successively. The primary structure analysis was determined by HPAEC-PAD, HPSEC, FT-IR, GC–MS, and NMR. The results showed p-LBP was a homogeneous heteropolysaccharide as a pectin molecule with an average molecular weight of 64 kDa p-LBP was an approximately 87 nm hollow sphere in 0.05 mol/L sodium sulfate solution determined by HPSEC-MALLS, DLS and TEM. A discussion of degradation patterns gave the detailed structural information of p-LBP. Therefore, the results from degraded fragments elucidated that the backbone of p-LBP was formed by → 4-α-GalpA-(1 →, repeatedly. Partial region was connected by → 4-α-GalpA-(1 → and → 2-α-Rhap-(1 →, alternatively. On the C-4 of partial → 2-α-Rhap-(1 → residues existed branches forming by → 4-β-Galp-(1 →, → 3-β-Galp-(1 → or → 5-α-Araf-(1 →, while on the C-6 of partial → 3-β-Galp-(1 → residues existed secondary branches forming by terminal-α-Araf, terminal-β-Galp or → 3-α-Araf-(1 →.

We have previously constructed an engineered anti-diabetic fusion protein using glucagon-like peptide-1 and the globular domain of adiponectin. Herein, we evaluated the therapeutic effects of this fusion protein (GAD) on high-fat diet (HFD)-fed ApoE−/− mice. The lipid-lowering effect of GAD was determined in C57BL/6 mice using a lipid tolerance test. The effects of GAD on HFD-induced glucose intolerance, atherosclerosis, and hepatic steatosis were evaluated in HFD-fed ApoE−/− mice using glucose tolerance test, histological examinations and real-time quantitative PCR. The anti-inflammation activity of GAD was assessed in vitro on macrophages. GAD improved lipid metabolism in C57BL/6 mice. GAD treatment alleviated glucose intolerance, reduced blood lipid level, and attenuated atherosclerotic lesion in HFD-fed ApoE−/− mice, which was associated with a repressed macrophage infiltration in the vessel wall. GAD treatment also blocked hepatic macrophage infiltration and prevented hepatic inflammation. GAD suppressed lipopolysaccharide-triggered inflammation responses on macrophages, which can be abolished by H89, an inhibitor of protein kinase A. These findings demonstrate that GAD is able to generate a variety of metabolic benefits in HFD-fed ApoE−/− mice and indicate that this engineered fusion protein is a promising lead structure for anti-atherosclerosis drug discovery.

Translational efficiency in Escherichia coli is strongly influenced by mRNA secondary structure of translational initiation region (TIR). We have previously reported that the expression of heterologous protein is directly related to the minimal folding free energy (ΔG) of the local secondary structure. However, identifying biologically relevant maximum and minimum levels of expression, or exploring the optimal level between them, is a key to successful optimization of heterologous protein expression. To systematically search a large range of the ΔG of TIR, we now present a quantitative analysis of the relationship between expression level and these ΔGs. The ΔG of TIR in green fluorescent protein is found to be linearly correlated with the fluorescence intensity over a range of tenfold change. The result demonstrates that the increasing ΔG of TIR can enhance the expression level linearly with no threshold or plateau.

To prepare recombinant tPep-(vascular endothelial growth factor) VEGF-B and assess its biological activity.This new VEGF fusion protein was constructed using a targeting peptide and prepared using E.coli. The tPep-VEGF-B was refolded from inclusion bodies and purified using affinity chromatography. Its bioactivity was determined in vitro using proliferation assay and wounding healing assay, and in vivo in zebrafish. By using the optimized downstream process, recombinant tPep-VEGF-B can be obtained with a purity of >90 % and a yield of 80 mg protein/l culture medium. The refolded protein is highly effective in promoting cell migration in vitro and in enhancing angiogenesis in vivo.We have constructed a new VEGF fusion protein with potential therapeutic application in treating metabolic diseases.

•A method is utilized for immobilizing natural polysaccharide as affinity absorbent.•An optimal solution of the synthesis of YCP-Sepharose is found.•The ligand density of YCP-Sepharose is impressive.•TLR2 and TLR4 are verified as receptors of YCP in B cells.YCP, an α-glucan from the mycelium of marine filamentous fungus Phoma herbarum YS4108, has great antitumor potential via enhancement of host immune through Toll-like receptor (TLR) 2 and TLR4 signaling. In the current study, YCP was coupled to EAH Sepharose 4B agarose beads to prepare the YCP-Sepharose affinity absorbent using 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) as the activating agent. An orthogonal experiment L9 (3)4 was applied to optimize the coupling procedure, giving the optimal parameters as follows: molar ratio of CDAP to YCP of 1:2, CDAP-activation time of 5 min, gel volume of 0.1 mL, and gel-incubation time of 72 h, respectively. Scanning electron microscopy analysis indicated successfully preparation of YCP immobilized sepharose beads, while these beads essentially maintained biological properties of free YCP since they can interact with TLR2 and TLR4 specifically at comparable level. Collectively, our findings provide an alternative approach to immobilize carbohydrate-based molecules for studying the carbohydrate–protein interaction.

•A miniature library of homogenous low-molecular-weight α-glucans was obtained.•We studied the molecular size-immunomodulating activity relationship of α-glucans.•We have indicated for the first time the probable functional unit of YCP, a marine α-glucan.YCP, an α-d-glucan with molecular weight of 2.4 × 103 kDa, was isolated from the mycelium of marine fungus Phoma herbarum YS4108. It interacted with TLR2 and TLR4 to induce B cells proliferation and activation. Here, a series of homogenous LMWYCPs (namely LMWYCP-1 to LMWYCP-6) were obtained by acid degradation. LMWYCP-3 and LMWYCP-4 were capable of inducing the proliferation of B cells dramatically. Further research involved TLR defunctionalization and competitive binding assay demonstrated that the immunomodulating activities of LMWYCP-4 was TLR4-dependent but TLR2-independent, while that mediated by LMWYCP-3 was neither TLR2- nor TLR4-dependent. Together with the structural information that LMWYCPs shared the similar structure features with YCP, we deduced that LMWYCP-4 may be the functional unit of YCP responsible for YCP/TLR4 interaction. This is the first time that the probable functional unit of glucan with the structure of α-d-(1–4)-linked residues in main chain and α-d-(1–6)-linked residues in branches is reported.

An acidic polysaccharide (SGP-2), with a molecular weight of 1880 kDa, was purified from the defatted whole-plant of Sarcandra glabra (Thunb.) Nakai. SGP-2 is mainly composed of glucose, galactose, mannose, arabinose and galacturonic acid in a molar ratio of 12.19:8.68:6.03:1.00:15.24. The primary structure analysis reveals that SGP-2 consists of 1,4-linked α-D-galacturonic acid, methyl-esterified 1,4-linked α-D-galacturonic acid, 1,5-linked α-L-arabinose, 1,4-linked α-D-mannose, 1,6-linked β-D-glucose and 1,3-linked β-D-galactose with branch chains of 1,4,6-linked β-D-glucose, 1,3,6-linked α-D-mannose and 1,4,6-linked α-D-galactose. The results of a cell viability assay and colony formation assay indicate that SGP-2 has a potent anti-proliferation activity on human osteosarcoma MG-63 cells. SGP-2 increases the proportion of apoptotic cells and activates caspase-3. In addition, the anti-proliferation effect induced by SGP-2 is blocked by the pan-caspase inhibitor. Moreover, SGP-2 inhibits the migratory capacity of MG-63 cells accompanied with the inhibition of receptor for advanced glycation end-products (RAGE) and nuclear factor-kappa B (NF-κB). Taken together, these results suggest that SGP-2 has anti-cancer potential in the treatment of human osteosarcoma.

•A novel acidic polysaccharide LBP-s-1 was obtained from Lycium barbarum L.•LBP-s-1 was of significant hypoglycemic effects and insulin-sensitizing activity.•LBP-s-1 promoted β cell proliferation and increased glucose metabolism.•LBP-s-1 increased the activity of hexokinase and pyruvate kinase.•Preliminary mechanisms of LBP-s-1 with marked hypoglycemic effects were elucidated.Diabetes mellitus is a group of complicated metabolic disorders characterized by high blood glucose level and inappropriate insulin secreting capacity due to decreased glucose metabolism and pancreatic β cell mass or dysfunction of β cells. Thus, improving glucose metabolism and preserving β cell mass and function might be useful for the treatment of diabetes. In this study, a novel acidic polysaccharide LBP-s-1 extracted from Lycium barbarum L. was obtained by purification using macroporous resin and ion-exchanged column. Monosaccharide composition analysis indicated that LBP-s-1 was comprised of rhamnose, arabinose, xylose, mannose, glucose, galactose, galacturonic acid in the molar ratio of 1.00:8.34:1.25:1.26:1.91:7.05:15.28. The preliminary structure features of LBP-s-1 were investigated by FT-IR, 1H NMR and 13C NMR. In vitro and in vivo hypoglycemic experiments showed that LBP-s-1 had significant hypoglycemic effects and insulin-sensitizing activity through increasing glucose metabolism and insulin secretion and promoting pancreatic β cell proliferation. Preliminary mechanisms were also elucidated.

A novel heteropolysaccharide (GPP-TL) was isolated from tetraploid Gynostemma pentaphyllum (Makino) leaf by hot water extraction and anion-exchange and gel permeation chromatography approaches. GPP-TL had a molecular weight of 9.3 × 103 Da and was primarily composed of glucose, galactose, and arabinose, with a molar ratio of 43:5:1, respectively. The chemical structure of GPP-TL was characterized using chemical and instrumental analyses. The results indicated the presence of (1→4)-α-d-glucopyranosyl, (1→4)-β-d-galactopyranosyl, (1→4,6)-linked-α-d-glucopyranosyl, and terminal 1→)-α-d-glucopyranosyl moieties in a molar ratio of 5.7:1:1.5:1, respectively. The results indicated that GPP-TL had glucose and galactose residues in the main chain with (1→6)-linked branches at glucose residues. In addition, GPP-TL exhibited scavenging capacities against hydroxyl, peroxyl, and DPPH radicals in vitro and had a stronger bile acid-binding ability than psyllium on a same-weight basis.

Globular adiponectin (GAD) as the active domain of adiponectin is a promising candidate for anti-diabetic drug development. The recombinant production of GAD in Escherichia coli, however, is difficult because it is mainly expressed as inclusion bodies which need to be refolded to regain function. In this study we developed a novel method for refolding of GAD with a high efficiency by using polyethylene glycol (PEG) conjugation. An artificially designed DNA sequence encoding for GAD was synthesized and inserted into the pET28a vector to construct an expression plasmid which was thereafter transformed into E. coli BL21 (DE3) host cells for heterologous expression. After bacterial cell culture employing auto-induction medium, the inclusion bodies were collected, washed and dissolved in guanidine hydrochloride before PEG conjugation. Then the PEG-conjugated GAD was refolded by dialysis and purified by two steps of chromatography. The refolded conjugate showed a marked glucose-lowering activity in mice, demonstrating that it had been successfully refolded. As a convenient method, PEGylation-aided refolding could also be tested on other proteins to explore its suitability.

Granulocyte colony-stimulating factor (G-CSF) is a cytokine widely used in cancer patients receiving high doses of chemotherapeutic drugs to prevent the chemotherapy-induced suppression of white blood cells. The production of recombinant G-CSF should be increased to meet the increasing market demand. This study aims to model and optimize the carbon source of auto-induction medium to enhance G-CSF production using artificial neural networks coupled with genetic algorithm. In this approach, artificial neural networks served as bioprocess modeling tools, and genetic algorithm (GA) was applied to optimize the established artificial neural network models. Two artificial neural network models were constructed: the back-propagation (BP) network and the radial basis function (RBF) network. The root mean square error, coefficient of determination, and standard error of prediction of the BP model were 0.0375, 0.959, and 8.49 %, respectively, whereas those of the RBF model were 0.0257, 0.980, and 5.82 %, respectively. These values indicated that the RBF model possessed higher fitness and prediction accuracy than the BP model. Under the optimized auto-induction medium, the predicted maximum G-CSF yield by the BP-GA approach was 71.66 %, whereas that by the RBF-GA approach was 75.17 %. These predicted values are in agreement with the experimental results, with 72.4 and 76.014 % for the BP-GA and RBF-GA models, respectively. These results suggest that RBF-GA is superior to BP-GA. The developed approach in this study may be helpful in modeling and optimizing other multivariable, non-linear, and time-variant bioprocesses.

Response surface methodology (RSM) was employed to optimize the parameters for polysaccharide extracted from the aerial parts of Sarcandra glabra (SGP). The optimum conditions were predicted as follows, ratio of water to raw material at 30, extracting temperature at 85 °C, extracting duration at 3 h, and yield was estimated at 4.55%. The experimental yield of SGP under the optimum conditions was 4.49 ± 0.09%. A homogenous polysaccharide (SGP-1) was obtained by purification using DEAE-cellulose-52 and Sephacryl S-400 column chromatography. SGP-1 showed a single symmetrical peak in high performance size-exclusion chromatography (HPSEC) and the average molecular weight (Mw) was estimated to be 1.06 × 104 Da. It was composed of glucose, galactose, and mannose in a ratio of 8.38:3.13:1 determined by gas chromatography (GC). The in vitro antioxidant tests showed that SGP-1 has significant inhibition effects on hydroxyl, superoxide anion, DPPH, ABTS radicals in a dose-dependent manner. This study indicated that SGP-1 could be used as a potential natural antioxidant.Highlights► A novel crude polysaccharide was extracted from Sarcandra glabra. ► The preliminary structure of SGP-1 was determined. ► The antioxidant activity of SGP-1 indicated it could be further explored.

Growing evidence suggests that type 2 diabetes mellitus (DM) is associated with age-dependent Alzheimer’s disease (AD), the latter of which has even been considered as type 3 diabetes. Several physiopathological features including hyperglycemia, oxidative stress, and dysfunctional insulin signaling relate DM to AD. In this study, high glucose-, oxidative stress-induced neuronal injury and intracerebroventricular-streptozotocin (ICV-STZ) animals as the possible models for diabetes-related AD were employed to investigate the effects of exendin-4 (Ex-4), a long-acting glucagon-like peptide-1 (GLP-1) receptor agonist, on diabetes-associated Alzheimer-like changes as well as the molecular mechanisms involved. Our study demonstrated that GLP-1/Ex-4 could exert a protective effect against reduced viability of PC12 cells caused by high glucose and that this protective effect was mediated via the PI3-kinase pathway. In addition, GLP-1/Ex-4 ameliorated oxidative stress-induced injury in PC12 cells. In rat models, bilateral ICV-STZ administration was used to produce impaired insulin signaling in the brain. Fourteen days following ICV-STZ injection, rats treated with twice-daily Ex-4 had better learning and memory performance in the Morris water maze test compared with rats treated with saline. Additionally, histopathological evaluation confirmed the protective effects of Ex-4 treatment on hippocampal neurons against degeneration. Furthermore, we demonstrated that Ex-4 reversed ICV-STZ-induced tau hyperphosphorylation through downregulation of GSK-3β activity, a key kinase in both DM and AD. Our findings suggests that Ex-4 can protect neurons from diabetes-associated glucose metabolic dysregulation insults in vitro and from ICV-STZ insult in vivo, and that Ex-4 may prove of therapeutic value in the treatment of AD especially DM-related AD.

Glucagon-like peptide-1 as an endogenous glucose-lowering peptide is a promising candidate for anti-diabetic drug development. Here, we developed a convenient method by coupling of refolding and intein-mediated self-cleavage (CRIS) to improve the recombinant production of a mutated glucagon-like peptide-1 (mGLP-1). Bacterial cell culture employing auto-induction was performed at 37 °C to avoid the intracellular self-cleavage of the intein fusion protein. The impacts of urea, pH, and temperature on the efficiency of CRIS were tested, and then, the optimized CRIS was established. Using the optimized method, we obtained the purified mGLP-1 with a yield of 3.41 mg peptide/g bacterial cells which was 5.6-fold higher than before. After that, using chromatography, peptide electrophoresis, and mass spectrometry, we determined the purity and molecular weight of the purified peptide and then confirmed its glucose-lowering activity by performing glucose tolerance test in mice. These results suggest that CRIS is a relatively simple and efficacious method for the recombinant production of mGLP-1, and as a general method, it can also be used for the recombinant preparation of some other proteins and peptides.

The Astragalus polysaccharide (APS) was extracted and purified from the roots of Astragalus membranaceus, and characterised for its chemical structure and potential health properties. The APS was composed of α-d-glc residues with the estimated equivalent dextran molecular weight of 2.07 × 104 Da. Periodate oxidation analysis, 1D and 2D NMR spectroscopy demonstrated that APS has repeating (1 → 4)-linked backbone with a (1 → 6)-linked branch every 10 residues. The APS possessed scavenging activities against hydroxyl radicals and hydrogen peroxide, and showed chelating effect on ferrous ions. The APS was also able to bind cholic and chenodeoxycholic acids in vitro. In addition, APS was able to stimulate activity of purified mouse B cells without promoting T cell proliferation. These data provided information for future development of APS as a nutraceutical.Highlights► APS is composed of glucose with (1 → 4)-linked backbone and (1 → 6)-linked branches. ► APS has a remarkable antioxidant activity in vitro. ► APS has strong ability of bile acid-binding in vitro. ► APS has a specific effect on proliferation of purified mouse B cells in splenocytes.

A domain at the NH2 terminal (N-terminal) of tumor necrosis factor receptor (TNFR) termed the pre-ligand binding assembly domain (PLAD). The finding that PLAD can mediate a selective TNFR assembly in previously researches provides a novel target to the prevention of TNFR signaling in immune-mediated inflammatory diseases (IMID). In this study, a natural N-terminal TNFR1 PLAD was obtained for the first time through the methods of GST-tag fusion protein expression and enterokinase cleavage. After purification with a Q Sepharose Fast Flow column, a natural N-terminal TNFR1 PLAD which purity was up to 95%, was obtained and was identified using Nano LC-ECI-MS/MS. Secondary structure analysis of PLAD was carried out using circular dichroism spectra (CD). After that, the TNFR1 PLAD in vitro anti-TNFα activity and the specific TNFR1 affinity were determined. The results proved that the natural N-terminal TNFR1 PLAD can selectively inhibit TNFα bioactivity mainly through TNFR1. It infers an effective and safe strategy for treating variety of IMID with a low risk of side effects in future.

A hydroxypropyl Ganoderma lucidum polysaccharide (H-GLP) was prepared from a low-value water-insoluble polysaccharide from G. lucidum (GLP) using propylene oxide under an alkaline condition. The H-GLP was characterised for its chemical structure with IR and 13C NMR spectra and analysed for its mono-sugar composition, molecular weight, and hydroxypropyl content. H-GLP contained mannose, glucose, and galactose in a mole ratio of 1.0:36.5:3.59, respectively, with an average molecular weight of 788 kDa and a hydroxypropyl content of 12.05%. H-GLP also had an excellent water solubility of more than 50 mg/mL, suggesting that hydroxypropylation might serve as an effective approach to enhance water solubility of GLP. H-GLP was also compared to the original GLP and ascorbic acid for antioxidant properties. H-GLP showed much stronger free radical scavenging capacity against hydroxyl and superoxide anion radicals and hydrogen peroxide than GLP. These results suggested the potential of hydroxypropylation in developing water-soluble antioxidative polysaccharides from GLP to enhance the profitability of G. lucidum production and processing industries.

Glucagon-like peptide-1 (GLP-1) is attracting increasing interest on account of its prominent benefits in type 2 diabetes. However, its clinical application is limited because of short biological half-life. This study was designed to produce a C-terminal site-specific PEGylated analog of cysteine-mutated GLP-1 (cGLP-1) to prolong its action. The gene of cGLP-1 was inserted into pET32a to construct a thioredoxinA fusion protein. After expression in BL21 (DE3) strain, the fusion protein was purified with Ni-affinity chromatography and then was PEGylated with methoxy-polyethylene glycol-maleimide (mPEG10K-MAL). The PEGylated fusion protein was purified with anion exchange chromatography and then was cleaved by enterokinase. The digested product was further purified with reverse-phase chromatography. Finally, 8.7 mg mPEG10K–cGLP-1 with a purity of up to 98% was obtained from the original 500 ml culture. The circular dichroism spectra indicated that mPEG10K–cGLP-1 maintained the secondary structure of native GLP-1. As compared with that of native GLP-1, the plasma glucose lowering activity of mPEG10K–cGLP-1 was significantly extended. These results suggest that our method will be useful in obtaining a large quantity of mPEG10K–cGLP-1 for further study and mPEG10K–cGLP-1 might find a role in the therapy of type 2 diabetes through C-terminal site-specific PEGylation.

As a promising type 2 anti-diabetic agent, glucagon-like peptide-1 (GLP-1) is attracting more and more interest. Mutated GLP-1 (mGLP-1) is an analog of native GLP-1. To facilitate the production and purification of mGLP-1, auto-induction and on-column cleavage was employed in this study. By using auto-induction system, after 24 h of shaking culture, about 12.6 g wet bacterial cells could be obtained from 1 l medium, and this was about 3.6 times more than that of the IPTG-induction group. After disruption and centrifugation, the fusion protein was directly purified and cleaved on Ni–Sepharose 6 Fast Flow column. Then, RESOURCE15 RPC column was used for further purification. By using these two steps of purification, about 1.58 mg of mGLP-1 with the purity of up to 98% could be obtained from 1 g wet bacterial cells. In the bioactivity study, mGLP-1 displayed a significant and dose-dependent glucose-lowering activity. These results suggested that auto-induction and on-column cleavage could facilitate the production and purification of mGLP-1. These methods could also be applied to the preparation of other proteins and peptides.

The chemical carboxylmethylated polysaccharide (C-GLP), which derived from water-insoluble crude Ganoderma lucidum polysaccharide (GLP), was prepared. Water solubility, chemical characterization, and antioxidant activities in vitro of C-GLP were determined. The solubility of C-GLP in distilled water reached 100 mg/ml, which was much higher than the solubility of GLP. Chemical analysis indicated that C-GLP was composed of Glc:Man:Gal = 33.0:1.0:3.4 with a molecular weight of 1.8 × 106 Da and a carboxymethyl content of 11.07%. The signals of carboxymethyl were found in IR and 13C NMR spectra. Moreover, a high antioxidant activity of C-GLP was observed, especially in scavenging of hydroxyl radical (83.7% at 5 mg/ml) and hydrogen peroxide (51.6% at 10 mg/ml). This study indicates the effects of carboxymethylation on water-insoluble polysaccharide and explores a potential antioxidant in food industry and pharmaceuticals.

In the present study, an aqueous extract from Choerospondias axillaries fruit was evaluated for its in vivo antioxidant activity, using the d-galactose induced mouse aging model, and for its in vitro scavenging effects on the superoxide anions, DPPH, H2O2, OH. The reducing power and Fe2+-chelating ability, as well as the inhibition of lipid peroxidation were also evaluated. The flavonoid and phenolic contents of the extract were determined. Pertaining to the in vivo activity, the intragastric administration of the extract inhibited d-galactose induced oxidative damage. Furthermore, in the in vitro assays, the extract showed a high antioxidant effect, especially scavenging of DPPH anions and its reducing power. The total content of phenolic and flavonoid compounds was 568 mg of gallic acid equivalents/g dry material and 2.09 mg of quercetin equivalents/g dry material respectively. These results provide scientific support for the empirical use of C. axillaries fruit as a medicine for cardiovascular diseases.

An activity-directed fractionation and purification process was used to identify the acetylcholinesterase inhibitory-active components of Rhodiola rosea L. (RR). Dried rhizome of RR was extracted with boiled ethanol. After removal of tannins, the extract was separated into chloroform, ethyl acetate, n-butanol and water fractions. Among these, chloroform and n-butanol fractions showed stronger activity by bioassay for anti-cholinesterase activity than did ethyl acetate and water fractions. The chloroform fraction was then subjected to separation and purification using silica gel column chromatography and Sephadex LH-20 chromatography. One compound, showing strong anti-cholinesterase activity, was identified by spectral methods (NMR, UV and MS) and by comparison with authentic samples. It proved to be hydroquinone.

•A novel lncRNA, 1700040D17Rik, was found to be down-regulated in EAE model.•After the treatment of rhIL23R-CHR, 1700040D17Rik was significantly increased.•1700040D17Rik was revealed to associate with the differentiation of Th17 cells.•1700040D17Rik was confirmed to regulate RORγt to affect Th17 cells.IL-23/STAT3 signaling pathway is a key process in Th17 cell differentiation, and Th17 cells are closely related to the development of autoimmune diseases. We previously designed and prepared rhIL23R-CHR protein to antagonize endogenous IL-23, showing effectiveness in the treatment of experimental autoimmune encephalomyelitis (EAE) in mice. To further elucidate the mechanism of action, mouse lncRNA microarray was used to screen expression profiles of lncRNAs, and a particular lncRNA, 1700040D17Rik was found to down-regulate in EAE model and its expression was significantly increased after the treatment by rhIL23R-CHR. The function of 1700040D17Rik was revealed to associate with the differentiation of Th17 cells through the regulation of the key transcription factor RORγt. Together, regulation of Th17 cells through lncRNA is responsible for the effects of rhIL23R-CHR to balance the immune responses, and 1700040D17Rik has the potential to serve as a therapeutic target or a biomarker for autoimmune diseases.

•Different linker peptides were introduced into the fusion protein construction.•The optimized fusion protein showed high potency in receptors activation.•The optimized fusion protein showed high potency in glucose- and lipid-lowering.Protein engineering has been successfully applied in protein drug discovery. Using this technology, we previously have constructed a fusion protein by linking the globular domain of adiponectin to the C-terminus of a glucagon-like peptide-1 (GLP-1) analog. Herein, to further improve its bioactivity, we reconstructed this fusion protein by introducing linker peptides of different length and flexibility. The reconstructed fusion proteins were overexpressed in Escherichia coli and purified using nickel affinity chromatography. Their agonist activity towards receptors of GLP-1 and adiponectin were assessed in vitro by using luciferase assay and AMP-activated protein kinase (AMPK) immunoblotting, respectively. The effects of the selected fusion protein on glucose and lipid metabolism were evaluated in mice. The fusion protein reconstructed using a linker peptide of AMGPSSGAPGGGGS showed high potency in activating GLP-1 receptor and triggering AMPK phosphorylation via activating the adiponectin receptor. Remarkably, the optimized fusion protein was highly effective in lowering blood glucose and lipids in mice. Collectively, these findings demonstrate that the bioactivity of this GLP-1 fusion protein can be significantly promoted by linker engineering, and indicate that the optimized GLP-1 fusion protein is a promising lead structure for anti-diabetic drug discovery.Download full-size image

•We report a soluble receptor, tIL12rβ1 for the treatment of lupus nephritis.•We present a new therapeutic strategy targeting IL-12/IL-23 p40 for lupus nephritis.•Both Th1 and Th17 cells are involved in the pathogenesis of cGVHD-induced SLE.Th1 and Th17 cells have been strongly implicated in the pathogenesis of systemic lupus erythematosus (SLE). Interleukin (IL)-12 and IL-23 respectively drive the polarization of Th1 and Th17 cells and share a common p40 subunit. In this study, the protective and therapeutic effects of a truncated human IL-12rβ1 receptor (tIL12rβ1) targeting IL-12/IL-23 p40 were evaluated in chronic graft-versus-host disease (cGVHD)-induced SLE-like model. The results indicated that tIL12rβ1 treatment effectively delayed the proteinuria onset and induced a significant remission of proteinuria, autoantibody production, and immune complex deposition in the mouse model. Remarkably, the therapeutic effects of tIL12rβ1 were predominantly dependent on the suppression of pathogenic Th1 and Th17 cell commitment through the reduction of RORγt and T-bet expression. Collectively, this receptor molecule may offer a new treatment option for SLE.

•The difference between glucose–BSA and glycoaldehyde–BSA: effects on PC12 cells.•The protective effect mediated by GLP-1 receptor on AGE-induced neurotoxicity.•GLP-1 can reduce cell tau phosphorylation induced by high glucose or glucose–BSA.•GLP-1 regulated tau phosphorylation through a signaling pathway involving GSK-3β.We have previously demonstrated that glucagon-like peptide-1 (GLP-1) receptor agonist ameliorated neurodegenerative changes in rat models of diabetes-related Alzheimer’s disease (AD), and protected neurons from glucose toxicity in vitro. Herein, we investigated the effects of GLP-1 receptor mediates on cell toxicity and tau hyperphosphorylation induced by advanced glycation end products (AGEs), which are associated with glucose toxicity, and the molecular mechanism in PC12 cells and the primary hippocampal neurons. Our study demonstrated that the similar protection effects of GLP-1 existed in PC12 cells treated with glucose–bovine serum albumin (BSA) in hyperglycemic conditions or with glycoaldehyde–BSA alone. Additionally, glucose–BSA alone did not induce significant cytotoxicity in PC12 cells, but resulted in tau hyperphosphorylation in primary hippocampal neurons in 24 h. And we found that GLP-1 could reduce cell tau phosphorylation induced by high glucose or glucose–BSA. Furthermore, our data in the present study suggested that GLP-1 regulated tau phosphorylation induced by AGEs through a signaling pathway involving glycogen synthase kinase 3β (GSK-3β), similarly to the GSK-3β inhibitor, lithium chloride. Our findings suggest that GLP-1 can protect neurons from diabetes-associated AGE insults in vitro, and provide new evidence for a potential therapeutic value of GLP-1 receptor agonist in the treatment of AD especially diabetes-related AD.

We describe an E. coli-based optimized system for the production of uricase with keto functional groups incorporated efficiently and site-specifically. In the process, the orthogonal suppressor tRNA/aminoacyl-tRNA synthetase (aaRS) pair specific for p-acetylphenylalanine (pAcF) was optimized to be effective at pAcF incorporation, showing no toxicity to the host cells. The efficiency of pAcF incorporation was further improved by coupling five copies of the T-stem mutant suppressor tRNA gene omitted the 3′ terminal CCA with two constitutive copies of the D286R mutant aaRS gene in a single-plasmid construct. To assay the utility of the optimized system, we incorporated pAcF in response to three independent amber nonsense codons (Lys21TAG, Phe170TAG, Lys248TAG) into uricase. Under optimized expression conditions, 24 mg/L mutant uricase was produced, corresponding to 40% of the yield of wild-type uricase (UOXWT). The desired specificity for incorporation of pAcF into uricase was confirmed. Kinetic measurements and spectroscopic study performed by CD did not show any relevant differences in the substrate affinity, the catalytic activity and protein secondary structure between native and mutant uricase. Additionally, the mutant uricase was site-specifically modified with methoxy-PEG-oxyamine (mPEG5K-ONH2). This efficient system provides reactive handles for a rational PEGylation to manipulate uricase structure and function and will be beneficial for enhancing the incorporation of other unnatural amino acids into proteins.Graphical abstract. A p15A-based single-plasmid system compatible with most E. coli expression vectors and strains was developed for incorporating p-acetylphenylalanine into uricase at the desired sites with high efficiency and fidelity. Under optimized expression conditions, 24 mg/L mutant uricase was produced, corresponding to 40% of the yield of wild-type uricase. Full-length mutant uricase was only produced in our optimized system when all three orthogonal components were present. This efficient system provides reactive handles for a rational PEGylation to manipulate uricase structure and function and will be beneficial for enhancing the incorporation of other unnatural amino acids into proteins.Download full-size imageHighlights► A suppressor tRNA was optimized to highly improve the efficiency of pAcF incorporation. ► The efficiency was further enhanced by optimizing suppressor tRNA/aaRS copy number. ► The suppressor tRNA/aaRS pair had no adverse effects on the growth of host cells. ► The versatility of pAC-pAcF produced high-yield of mutant uricase with full activity. ► The keto groups provide reactive handles for a rational PEGylation of uricase.

•De novo generation of antibody-secreting cells from naïve lymphocytes in vitro.•Convert in vitro immunization of unfractionated PBMCs to step-by-step cell culture.•Succeed to differentiate naïve CD4+ T cells to Tfh cells for in vitro immunization.•Detected AID and Blimp-1 indicate class-switch recombination might be initiated.In vitro immunization with antigens and cytokines triggers specific human B-cell response in short periods and is therefore superior to conventional in vivo immunization for antibody development. However, this new technology is limited by low efficiency, poor reproducibility, and requirement of pre-immunized lymphocytes. In this study, we demonstrate a novel method for de novo inducing antigen-specific human B cells in vitro. Unlike previous in vitro immunization of unfractionated PBMCs, we firstly optimized the conditions for inducing monocyte-derived dendritic cells (DCs) to efficiently capture, process, and present antigens. Instead of using the conventional method to activate Th2 cells for in vitro immunization, we succeeded to differentiate naïve CD4+ T cells into T follicular helper (Tfh) cells using antigen-sensitized DCs and cytokine cocktail. We discovered the differentiated T cells expressed ICOS, PD-1, BCL-6, and IL-21 at high levels. After 12 days of T-B co-culture, we observed induced T cells efficiently promoted naïve B cells to differentiate into plasmablasts secreting antigen-specific antibodies, with expression of Blimp-1 and AID related to affinity maturation and class switching. Thus, we established a new co-culture system with naïve lymphocyte populations for de novo acquisition of specifically in vitro immunized B cells potentially for development of therapeutic antibodies, which also provides novel insights into understanding the complex interactions among immune cells in lymph nodes.Download high-res image (184KB)Download full-size image