A codon-optimized Escherichia coli appA phytase gene was synthesized and expressed in Pichia pastoris. Two residue substitutions (Q258N, Q349N) were sequentially introduced to enhance its glycosylation activity. Secretion of appA-Q258N/Q349N was approx. 0.3 mg ml−1 and enzyme activity reached 1,030 U ml−1. Purified appA-Q258N/Q349N had a specific activity of 3,137 U mg−1 with an MW of approx. 53 kDa. Compared with appA-WT, appA-Q258N/Q349N showed over 40 % enhancement in thermostability (85 °C for 10 min) and 4–5 °C increases in the melting temperatures (Tm). The Km and Kcat of appA-Q258N/Q349N were 0.43 mM and 3,058 s−1, respectively, which are similar with that of appA-WT. The mutant appA-Q258N/Q349N obtained in this study could be used for the large-scale commercial production of phytase.

BmK CT, one of the key toxins in the venom of the scorpion, Buthus martensii Karsch, can interact specifically with glioma cells as a chloride channel blocker and inhibit the invasion and migration of those cells via MMP-2. A recombinant adenovirus, Ad-BmK CT, was constructed and characterized by in vitro and in vivo studies, using MTT cytotoxicity assay and the glioma C6/RFP (red fluorescence protein)/BALB/c allogeneic athymic nude mice model, respectively. The adenovirus-mediated expression of BmK CT displayed a high activity in suppressing rat C6 glioma cells growth and invasion thereby suggesting that this recombinant adenovirus may be a powerful method for treating glioblastoma.

Centrin is a low molecular mass (20 KDa) protein that belongs to the EF-hand superfamily. In this work, the interaction between the Tb3+-saturated C-terminal domain of Euplotes octocarinatus centrin (Tb2-C-EoCen) and 2-p-toluidinylnaphthalene-6-sulfonate (TNS) was investigated using difference UV–vis spectra and the fluorescence spectra methods. In 100 mM N-2-hydroxy-ethylpiperazine-N-2-ethanesulfonic acid (Hepes) at pH 7.4, with the addition of Tb2-C-EoCen, four new peaks were observed at 265 nm, 278 nm, 317 nm and 360 nm by absorptivity compared with blank solution of TNS. At the same time, the reaction could be measured by fluorescence spectra. The fluorescence emission of TNS was shifted from 480 nm to 445 nm in the presence of Tb2-C-EoCen. Meanwhile, its fluorescence intensity was increased markedly. The 1:1 stoichiometric ratio of C-EoCen to TNS was confirmed by fluorescence titration curves. The conditional binding constants of TNS with C-EoCen and Tb2-C-EoCen were calculated to be log K(C-EoCen-TNS)=5.32±0.04  M−1 and log K(Tb2-C-EoCen-TNS)=5.58±0.12 M−1, respectively. In addition, the protein of Tb2-C-EoCen binding with melittin was also studied. Based on the fluorescence titration curves, the 1:1 stoichiometric ratio of Tb2-C-EoCen to melittin was confirmed. And the conditional binding constant of C-EoCen with melittin was calculated to be log Ka′=6.79±0.17 M−1.Highlights► Tb3+ induced conformational changes of protein C-EoCen from closed state to open state. ► Conformational changes resulted in the exposure of hydrophobic surfaces on C-EoCen. ► Tb2-C-EoCen may bind with target peptide melittin.

The binding of both factors (eRF1 and eRF3) is essential for fast kinetics of the termination of protein translation. The C-terminal domain of eRF1 is known to interact with the C domain of eRF3. Eo-eRF1b contains two highly conserved tryptophan residues (W-11 and W-373), W-11 located in the Eo-eRF1b N domain and W-373 located in the EoeRF1b C domain. Fluorimetry was used to study the interactions of the proteins. When binding with Eo-eRF3Cm6, the emission peak of Eo-eRF1b is blue shifted, while the emission peak of Eo-eRF1bC has no notable change. Our results suggest that the eRF1-eRF3 interaction induces the N and C domain of eRF1b to become closer to each other.

The binding of Mg2+ with the Euplotes octocarinatus centrin (EoCen) and the effect of Mg2+ on the binding of EoCen with the peptide melittin were examined by spectroscopic methods. In this study, it was found that Mg2+ may bind with Ca2+-binding sites, at least partly, on EoCen, which displays ∼10-fold weaker affinity than Ca2+. In the presence of Mg2+, Ca2+-saturated EoCen undergoes significant conformational changes resulting in decreased exposure of hydrophobic surfaces on the protein. Additionally, excess Mg2+ did not change the stoichiometry, but rather reduced the affinity of EoCen to melittin. The Mg2+-dependent decrease in the affinities of EoCen to melittin is an intrinsic property of Mg2+, rather than a nonspecific ionic effect. The inhibitory effect of Mg2+ on the formation of complexes between EoCen and melittin may contribute to the specificity of EoCen in target activation in response to cellular Ca2+ concentration fluctuations.Highlights► Mg2+ may bind with EoCen and located, at least partially, within the calcium-binding sites. ► Mg2+ plays an opposite role to the Ca2+-induced conformations changes for EoCen. ► Mg2+ decreased the affinities of mimic peptide melittin with EoCen.

An Buthus martensii Karsch Insect Toxin (BmK IT) gene was inserted into the genome of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) to construct a recombinant baculovirus, AcMNPV-BmK IT. The expression of BmK IT was confirmed using RT-PCR, dot blot and SDS-PAGE analysis. Dose-lethal time responses to Spodoptera exigua larvae were compared between wild-type baculovirus AcMNPV and recombinant virus AcMNPV-BmK IT. At the concentration of 1 × 107 PlBs/mL, the median lethal time of recombinant baculovirus (LT50 = 73.6 h) on third instar S. exigua larvae showed an improvement of 13.2% over the efficacy of wild type virus (LT50 = 84.8 h) during a 192 h infection.

We have evaluated the effects of the antibiotic hygromycin B on cotton (Gossypium hirsutum L.) callus induction, callus proliferation, and seed germination. Nontransgenic cotyledon and hypocotyl showed obvious variance in tolerance to hygromycin. Cotyledons were more sensitive to hygromycin than hypocotyls. Hygromycin at 7.5 and 20 mg l−1 completely inhibited callus initiation from cotyledon and hypocotyl explants, respectively. Nontransformed calli did not grow on media supplemented with 10 mg l−1 hygromycin and were killed at 15 mg l−1. In seed germination assay, the presence of 20 mg l−1 hygromycin significantly suppressed shoot and root elongation of seedlings. This hygromycin concentration was applied to select regenerated transgenic plantlets and their progenies. Based on these results, we developed an efficient hygromycin selection protocol for Agrobacterium-mediated cotton transformation and regeneration.

The low yield and poor folding efficiency in vivo of soluble and active recombinant cysteine-rich proteins expressed in Escherichia coli are a major challenge for large-scale protein production and purification. Expression vectors containing Buthus martensii Karsch insect toxin (BmK IT) fused to the C terminus of the intein Ssp DnaB were constructed in an attempt to overcome this problem. Following purification and intein self-cleavage, the fusion protein His6-intein-IT produced insoluble BmK IT, while intein-IT-His6 generated soluble and properly folded BmK IT. This result indicated that the positioning of the His6 tag has a key role in the production of soluble and functional BmK IT.

Termination of translation in eukaryotes requires two polypeptide chain-release factors, eRF1 and eRF3. eRF1 recognizes stop signals, whereas eRF3 is a ribosome-dependent and eRF1-dependent GTPase. Polypeptide release factor eRF3 consists of N-terminal variable region and C-terminal conserved part. C-terminal part of eRF3 is responsible for termination of the translation. In the present study, the C-terminal of Euplotes octocarinatus eRF3 (eRF3C) and truncate eRF3C lacking 76 amino acids in C-terminal (eRF3Ct) were expressed in Escherichia coli. The recombinant GST-eRF3C and GST-eRF3Ct polypeptides were purified by affinity chromatography using glutathione Sepharose 4B column. After enzymatic cleavage of GST tail, the eRF3C and eRF3Ct protein were obtained. Pull-down analysis showed that the recombinant GST-eRF3C and GST-eRF3Ct polypeptides interacted with E. octocarinatus polypeptide chain release factor eRF1a. This result suggested that the C-terminal of eRF3 having 76 amino acids were not required for the binding of eRF1a in Euplotes octocarinatus.

Chlorotoxin, one of the key toxins in scorpion Leiurus quinquestriatus venom, has been shown to bind specifically to glioma cell surface as a specific chloride channel blocker. In this study, a purified, recombinant chlorotoxin-like peptide from the scorpion Buthus martensii Karsch (named rBmK CTa) was characterized by in vivo and in vitro studies. The results from cell proliferation assay with human glioma (SHG-44) cells showed that rBmK CTa inhibits the growth of glioma cells in a dose-dependent manner, with an IC50 value of approximately 0.28 μM. Under the same conditions, the IC50 value for normal astrocytes increased to 8 μM. This clearly indicated that rBmK CTa had specific toxicity against glioma cells but not astrocytes. Results from whole-cell patch-clamp recording showed that chloride current in SHG-44 was inhibited by rBmK CTa in a voltage-dependent manner and percent inhibitions for the blocking action of rBmK CTa (0.07 and 0.14 μM) on ICl was 17.64 ± 3.06% and 55.86 ± 2.83%, respectively. Histological analysis of rBmK CTa treated mice showed that brain, leg muscle and cardiac muscle were the target organs of this toxin. These results suggest that rBmK CTa may have potential therapeutic application in clinical treatment of human glioma. It represents an approach for developing a novel therapeutic agent.