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| Prokaryotic Expression, Purification and Activity Study of Kunitz Type Serine Protease Inhibitor IsKuI-1 |
| CUI Hong-di1,2, SHAO Zheng1, DENG Li1, SITU Yong-li1, PENG Li-fei1,3 |
1. Department of Parasitology, Guangdong Medical College, Zhanjiang 524023, China;
2. Department of Pharmacology, Guangdong Medical College, Zhanjiang 524023, China;
3. Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, Dongguan 523808, China |
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Abstract Objective: To express and purify the Kunitz type serine protease inhibitor IsKuI-1 (GenBank NO. AAY66517) from E. coli, and analyze its anticoagulation and anti-protease activity. Methods: The prokaryotic expression plasmid pET32a-sumo/IsKuI-1 was constructed and then transferred into E. coli BL21 (DE3). The fusion protein Trx-SUMO-IsKuI-1 was expressed after inducing with IPTG and purified by Ni-NTA resin affinity chromatography. The fusion tag was cleaved with SUMO protease on the resin bed. Prothrombin time and activated partial thromboplastin time assay was used to detect the anticoagulant activity and a single stage chromogenic assay was used to determine the inhibitory activity against serine proteases. Results: rIsKuI-1 was obtained successfully using the prokaryotic expression system and showed no prolongation of clot time. rIsKuI-1 has potential inhibitory activity against human neutrophil elastase (IC50 =1.83 μmol/L), but no inhibition of human cathepsin G, human pancreatic chymotrypsin, human pancreatic trypsin, human chymase, bovine pancreatic α-chymotrypsin and porcine pancreatic trypsin. Conclusion: IsKuI-1 is an inhibitor of human neutrophil elastase. So it laid a foundation for the further investigation of the biological function and application of IsKuI-1.
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Received: 18 September 2014
Published: 25 December 2014
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