纤细齿梗孢丝氨酸蛋白酶cDNA克隆及其序列分析和表达

cDNA Cloning and Sequence Analysis of the Serine Protease from the Mycoparasite Fungus Olpitrichum tenellum and Its Expression in Pichia pastoris

纤细齿梗孢Olpitrichum tenellum是一种寄生在轮枝镰孢菌Fusarium verticillioides上的活体营养菌寄生真菌。丝氨酸蛋白酶在菌寄生过程可能起到重要作用。根据丝状真菌丝氨酸蛋白酶的同源保守序列设计简并引物,通过RT-PCR及RACE的方法,克隆得到1845bp的全长cDNA片段。其可读框为1554bp(GenBank登录号为EU368754),编码517个氨基酸的蛋白质。比对分析发现该蛋白是一种分泌型的丝氨酸蛋白酶,属于丝氨酸蛋白酶S8家族的枯草杆菌蛋白酶subtilisin,具有典型的信号肽-前肽-成熟肽的结构。将该基因可读框除去信号肽插入酵母表达载体pPIC9K,转化毕赤酵母Pichia pastoris GS115,在甲醇诱导下成功分泌出具有生物活性的重组蛋白酶,诱导120h后酶活性可达153U/ml。重组蛋白酶经DEAE-Sepharose层析进行纯化,SDS-PAGE分析其分子量为39kD。其反应的最适温度为60℃,最适pH为8。

Olpitrichum tenellum is a biotrophic mycoparasite fungus of Fusarium verticillioides.Serine protease of O.tenellum may play an important role in its mycoparasitic process.A cDNA that encodes an extracellular serine protease from O.tenellum was cloned,analyzed and expressed in Pichia pastoris.The degenerate primers based on conserved domains of other reported serine proteases were designed to obtain a cDNA fragment by RT-PCR and RACE.Full length of the cDNA is 1845 bp,with an ORF of 1554 bp encoding 517 amino acids,registered in GenBank with an accession number EU368754.Homologic analyses showed that the deduced protein is a kind of secretory subtilisin belonging to the serine protease family S8,with the typical structure of a signal peptide,a propeptide,a catalytic domain,a P/middle or homoB domain and a C-terminus.The ORF without the signal peptide was subcloned into the expression vector pPIC9K and expressed in P.pastoris GS115.The activity level of the recombinant protease reached to 153 U/ml after 120 h methanol induction.The recombinant protease was purified by DEAESepharose chromatography and its molecular weight was 39 kD determined by SDS-PAGE.The optimum pH and temperature for the activity of the recombinant protease were 8 and 60 ℃,respectively.