重组沙蚕溶栓活性蛋白酶的纯化及其性质研究  

Purification and characterization of recombinant protease with thrombolytic activity of Perinereis aibuhitensis Grube

作　　者：李荣贵[1] 赵峰[1] 杨宏[1] 杜桂彩[1] 汪靖超[1] 王斌[1] 

机构地区：[1]青岛大学生物系,山东青岛266071

出　　处：《中国海洋药物》2007年第2期1-6,共6页Chinese Journal of Marine Drugs

基　　金：国家重点基础研究前期专项基金资助项目(2004CCA02400)

摘　　要：目的表达、纯化重组沙蚕溶栓活性蛋白酶并对其性质进行研究。方法将表达栽体pMAL-PPA转化入E.coli DH5α构建工程菌,IPTG诱导工程茵大量表达麦芽糖结合蛋白-沙蚕溶栓活性蛋白酶(MBP- PPA),细胞裂解液中融合蛋白经Amylose-resin亲和层析与DEAE-Sepharose FF离子交换层析得到了纯化．用Factor Xa切割融合蛋白后经酪蛋白平板法检测其纤维蛋白溶酶原激活活性．然后对其部分性质进行了研究。结果构建了表达MBP—PPA的工程菌,经纯化得到相对分子质量约51kDa的融合蛋白,Factor Xa切割融合蛋白后,重组沙蚕溶栓活性蛋白酶(PPA)体外具有纤维蛋白溶酶原激活活性,性质研究表明PPA热稳定性好,最适pH为8.0,该酶在pH6.0～9.0范围内有较好的稳定性,酶活在有机溶剂中至少维持20d,25 mmol．L^(-1)PMSF能完全抑制其活性。结论证实PPA是一种纤溶酶原激活荆,且将来有望成为一种新型溶栓药物。Objective To express and purify recombinant protease with thrombolysis activity of Perinereis aibuhitensis Grube and study on its characterization. Methods pMAL-PPA was introduced into E. coli DH5α to construct engineering bacteria and overexpression of the protease of fused with maltose binding protein（MBP-PPA） was achieved with IPTG induction. The fusion protein was purified by affinity chromatography on amylose-resin column followed by chromatography on a DEAE-sepbarose FF column. PPA cut with Factor Xa was assayed using casein plates supplied with plasminogen. Results Engineering bacteria expressing maltose binding protein-thrombolytic protease of P. aibuhitensis were constructed and overexpression of MBP-PPA was achieved with IPTG induction. A recombinant fusion protein of 51kD was purified, and PPA cut down from the fusion protein had a plasminogen-actirating activity. The protease showed a good thermal stability with an optimal pH of 8, 0. This enzyme was also relatively stable in a pH range of 6.0-9.0 and still active after stored in organic solvents for 20d. Conclusion PPA was verified as a plasminogen activator, and might be a new thrombolytic medicine in the future.

关 键 词：沙蚕 溶栓活性蛋白酶 纯化 性质 

分 类 号：Q959.19[生物学—动物学] TQ460[化学工程—制药化工]