重组瑞替普酶在毕赤酵母中的表达

Expression of recombinant reteplase in Pichia pastoris

【目的】瑞替普酶(重组组织纤溶酶原激活物,rt PA)被认为是第三代安全有效的溶栓剂,以p PIC9K为载体,以3种不同表型的毕赤酵母(Pichia pastoris)为宿主,探索适合rt PA分泌型表达的最佳体系。【方法】以质粒p ET28a-rt PA为模板,设计特异性引物,PCR扩增目的基因rt PA,插入分泌型表达载体p PIC9K中,获得重组表达质粒p PIC9K-rt PA。重组质粒经限制性内切酶Sal I线性化后,电击转化至3种不同表型的P.pastoris(GS115、SMD1168、KM71)中进行组成型表达;重组表达体系进行甲醇诱导表达,对产物进行Western blot鉴定,并采用纤维蛋白平板溶圈法测定其活性。【结果】重组蛋白分子量约为43 k D;rt PA-GS115和rt PA-KM71均在39 k D处有特异性条带,且前者在32 k D处有轻微降解条带,而后者并无此现象;rt PA-SMD1168无降解现象,且rt PA-SMD1168比活性较rt PA-GS115高27%;rt PA-KM71表达量和活性均为最低。【结论】从重组蛋白生物活性出发,P.pastoris SD1168可作为rt PA的最佳表达体系,在控制宿主蛋白酶活性、减少产物降解的前提下,P.pastoris GS115也是rt PA表达的优选体系。

[Objective] Reteplase （rtPA, recombinant tissue plasminogen activator） is considered as the third generation of safe and effective thrombolytic agent. In order to explore the suitable system for secretory expression of rtPA, the rtPA gene was expressed in three different phenotypes of Pichia pastoris with pPIC9K as the vector. [Methods] The rtPA gene was obtained from plasmid pET28a-rtPA and inserted into the secretory expression vector pPIC9K. The recombinant plasmid pPIC9K-rtPA was digested with restriction endonuclease Sal I and then transformed into P. pastoris GS 115, SMD 1168 and KM71, respectively. The positive clones were screened on MD-G418 plates and verified by PCR. The expression of rtPA in the recombinant strains were induced with methanol, and analyzed by Western blot and fibrin agarose plate. [Results] The molecular weight of recombinant proteins was about 43 kD; both rtPA-GS115 and rtPA-KM71 had a specific band at 39 kD, and the former had a slight degradation band at 32 kD, while the latter did not; rtPA-SMD 1168 showed no degradation. The specific fibrinolytic activity of rtPA-SMD l 168 was 27% higher than that ofrtPA-GS115, while the expression and activity of rtPA-KM71 displayed the lowest level among tested samples. [Conclusion] According to the expression of fibrinolytic activity, P. pastoris SMD1168 was proven to be the best expression system. On the premise of controlling the activity of protease and reducing the degradation of products, P. pastoris GS 115 was selected as the preferred system for rtPA expression.