球孢白僵菌丝氨酸蛋白酶基因CDEP-1在毕赤酵母中的表达

我们从球孢白僵菌中克隆了丝氨酸蛋白酶Pr1类基因CDEP-1。为明确CDEP-1的功能、评价其在害虫生物防治中的潜力,需要大量制备具有生物活性的CDEP-1编码蛋白。由于大肠杆菌系统表达真核基因存在产物复性困难的问题,本文利用毕赤酵母系统来表达CDEP-1。结果表明,CDEP-1可在毕赤酵母中高效的分泌表达,而且产物活性高,甲醇诱导48h后上清液中的酶活即可达到38,266U/L。诱导表达的上清液经浓缩后进行凝胶过滤层析,得到了CDEP-1的初纯品,蛋白质含量为50mg/L。将纯化的蛋白酶CDEP-1免疫家兔,制备了CDEP-1的抗血清。Westernblotting分析表明,制备的抗血清可特异性地检测CDEP-1。

Preparation of a serine protease CDEP-1 of Beauveria bassiana by pichia pastoris expression system

CDEP-1, a serine protease, produced by entomopathogenic fungus Beauveria bassiana, is a potential virulence factor against insects. To meet the need of assessing the roles of CDEP-1 in the pest bio-control, large amount of active CDEP-1 was required. Escherichia coli is the microbial host widely used for the production of heterologous protein. However, gain of active proteins has remained a recalcitrant problem for the E. coli expression system due to being prone to form inclusion bodies. On the other hand, the methylotrophic yeast Pichia pastoris, which can perform the post-translational modifications of eukaryotic system, has turned out to be a suitable host organism for production of active protein. To produce large scale of active CDEP-1, the gene encoding CDEP-1 was cloned into yeast expression vector, pPIC9K, and then delivered into a Pichia pastoris strain, GS115. The recombinant CDEP-1 can be secreted into the culture medium in the recombinant Pichia pastoris, and the total enzyme activity reached 38, 266U/L at 48th h after inducing by methanol. The product of CDEP-1 was purified to homogeneity through gel filtration chromatography detected by SDS-PAGE analysis. Results demonstrated that the Pichia pastoris expression system is useful in producing recombinant active CDEP-1 (expression levels up to 50 mg/L). Additionally, with purified product, the rabbit antiserum againstCDEP-1 was prepared by immunization.