Kunitz型丝氨酸蛋白酶抑制剂IsKuI-1的原核表达、纯化及活性研究

目的:原核表达并制备重组蜱kunitz型丝氨酸蛋白酶抑制剂IsKuI-1,检测其抗凝血及抑制蛋白酶活性。方法:构建pET32a-sumo/IsKuI-1原核表达质粒,并转入到E. coli BL21(DE3)中,用IPTG诱导表达。表达产物经Ni-NTA亲和层析,在层析柱上用SUMO蛋白酶切割融合伴侣,纯化后得到重组目的多肽rIsKuI-1。用PT及aPTT法检测重组目的多肽的抗凝活性,发色底物法检测rIsKuI-1对丝氨酸蛋白酶的抑制活性。结果:用原核表达系统获得了rIsKuI-1,其无延长PT及aPTT活性,对人中性粒细胞弹性蛋白酶具有较好的抑制活性(IC50=1.83μM),且特异性强。结论:IsKuI-1是一种活性较好的人NE抑制剂。因此为进一步探讨rIsKuI-1的生物学功能及其作为新药开发应用奠定了基础。

Prokaryotic Expression,Purification and Activity Study of Kunitz Type Serine Protease Inhibitor IsKuI-1

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.