优化圆叶叶口蝠唾液纤溶酶原激活物α2（DSPAα2）在毕赤酵母中的基因合成、表达及活性纯化

Optimized gene synthesis, expression and purification of active salivary plasminogen activator alpha2 (DSPAalpha2) of Desmodus rotundus in Pichia pastoris.

作者信息

Wei Zhaorong, Wang Yueju, Li Gangqiang, Li Xiaokun, Liu Dehu

机构信息

Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, PR China.

出版信息

Protein Expr Purif. 2008 Jan;57(1):27-33. doi: 10.1016/j.pep.2007.09.001. Epub 2007 Sep 14.

DOI:10.1016/j.pep.2007.09.001

PMID:17950617

Abstract

Vampire bat salivary plasminogen activators (DSPAs) are thrombolytic agents that are under clinical investigation for the treatment of acute ischemic stroke. In this study, the synthetic active salivary plasminogen activator alpha2 (DSPAalpha2) gene optimized for the preferred codons of Pichia pastoris was assembled from 48 oligonucleotides, and cloned into the yeast expression vector pPIC9 with a strong enhancer from human cytomegalovirus (HCMV). This system achieved high expression of an active DSPAalpha2 in P. pastoris yeast GS115. Secreted active DSPAalpha2 recombinant protein was purified from broth supernatant by a simple one-step procedure on Sephadex chromatography and was confirmed by SDS-PAGE and Western blot analysis. ELISA showed that 2.5mg of recombinant protein could be obtained from 100-ml culture broth supernatant. The fibrinolytic activity of the recombinant DSPAalpha2 was 1.28 x 10(5)IU/mg.

摘要

吸血蝙蝠唾液纤溶酶原激活剂（DSPAs）是正在进行急性缺血性中风治疗临床研究的溶栓药物。在本研究中，针对毕赤酵母偏好密码子优化的合成活性唾液纤溶酶原激活剂α2（DSPAα2）基因由48条寡核苷酸组装而成，并克隆到带有来自人巨细胞病毒（HCMV）强增强子的酵母表达载体pPIC9中。该系统在毕赤酵母GS115中实现了活性DSPAα2的高表达。分泌的活性DSPAα2重组蛋白通过在Sephadex色谱上的简单一步法从肉汤上清液中纯化出来，并通过SDS-PAGE和Western印迹分析进行了确认。ELISA显示，从100毫升培养液上清液中可获得2.5毫克重组蛋白。重组DSPAα2的纤溶活性为1.28×10⁵IU/毫克。