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Zhou Q.,China Agricultural University | Xu W.,China Agricultural University | Zhu L.,China Agricultural University | Yu T.,China Agricultural University | And 4 more authors.
Analytical Letters | Year: 2013

Snake venom contains bioactive materials for drug development, diagnosis, and treatment. After separating and purifying the kallikrein-like enzyme (AHP-Ka) from Agkistrodon halys pallas venom for the first time, a monoclonal antibody against AHP-Ka was prepared and characterized. An indirect sandwich enzyme-linked immunosorbent assay (ELISA) based on the monoclonal antibody was developed and validated for the pharmacokinetic analysis of AHP-Ka in rat plasma. The method was calibrated using rat plasma and 1:100 dilution of plasma was selected to prepare a calibration curve to validate the precision, accuracy, and stability of the ELISA method. A good linear relationship was obtained in a working range from 3.9 ng/mL to 62.5 ng/mL with a limit of detection of 2.94 ng/mL. Intra- and inter-batch precision were less than 10%. The average recovery ranged from 94.6% to 104.4% in rat plasma at the concentrations of 5 ng/mL, 15 ng/mL, and 45 ng/mL, respectively. The ELISA method was successfully used for the pharmacokinetic study of AHP-Ka in Sprague-Dawley rat plasma after intravenous administration. The work is expected to contribute to future preclinical development of AHP-Ka. © 2013 Copyright Taylor and Francis Group, LLC.


Zhang Y.,China Agricultural University | Xu W.,China Agricultural University | Ma B.,China Agricultural University | Ma B.,Saisheng Pharmaceutical Company | And 5 more authors.
Journal of the Science of Food and Agriculture | Year: 2012

Background: Viper snake venoms contain a great variety of toxic proteins. These components mediate their toxicity by either stimulating or inhibiting the haemostatic system of human victims or experimental animals, resulting in common clinical complications of blood clotting or uncontrolled haemorrhage. Therefore it is deemed important to isolate the active component(s) from snake venom with kallikrein-like activity. Results: A kallikrein-like proteinase of Agkistrodon halys pallas snake venom, designated AHP-Ka, was purified by anion exchange chromatography and affinity chromatography. Physicochemical studies showed that the purified enzyme was a 34 kDa monomeric glycoprotein, the molecular weight of which decreased to 26 kDa after deglycosylation with peptide N-glycosidase F (PNGase F). Sequence studies on the NH 2-terminal region of the protein indicated that AHP-Ka shared a high degree of sequence homology with other serine proteinases from snake venoms. AHP-Ka showed high catalytic activity and kallikrein-like activity on substrates such as arginine esterase BAEE and chromogenic H-D-Pro-Phe-Arg-pNA·2HCl (S-2302) and was inhibited by protease inhibitor phenylmethylsulfonyl fluoride (PMSF). Conclusion: The results showed that AHP-Ka isolated from A. halys pallas snake venom and purified by anion exchange chromatography and affinity chromatography is in fact a kallikrein-like enzyme. © 2011 Society of Chemical Industry.


Wang Y.,Tianjin University | Wang Y.,China Agricultural University | Xu W.,China Agricultural University | Kou X.,Tianjin University | And 6 more authors.
Protein Expression and Purification | Year: 2012

Wheat germ cell-free protein synthesis systems have the potential to synthesize functional proteins safely and with high accuracy, but the poor energy supply and the instability of mRNA templates reduce the productivity of this system, which restricts its applications. In this report, phosphocreatine and pyruvate were added to the system to supply ATP as a secondary energy source. After comparing the protein yield, we found that phosphocreatine is more suitable for use in the wheat germ cell-free protein synthesis system. To stabilize the mRNA template, the plasmid vector, SP6 RNA polymerase, and Cu 2+ were optimized, and a wheat germ cell-free protein synthesis system with high yield and speed was established. When plasmid vector (30 ng/μl), SP6 RNA polymerase (15 U), phosphocreatine (25 mM), and Cu 2+ (5 mM) were added to the system and incubated at 26 °C for 16 h, the yield of venom kallikrein increased from 0.13 to 0.74 mg/ml. The specific activity of the recombinant protein was 1.3 U/mg, which is only slightly lower than the crude venom kallikrein (1.74 U/mg) due to the lack of the sugar chain. In this study, the yield of venom kallikrein was improved by optimizing the system, and a good foundation has been laid for industrial applications and for further studies. © 2012 Published by Elsevier Inc.

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