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.
出处
《中国海洋药物》
CAS
CSCD
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)
关键词
沙蚕
溶栓活性蛋白酶
纯化
性质
P. aibuhitensis
protease with thrombolytic activity
purification
characterization
