毕赤酵母分泌表达SAMS及表达产物活性分析

Secreted Expression and Biological Assay of SAMS in Pichia pastoris

[目的]利用毕赤酵母分泌表达S-腺苷甲硫氨酸合成酶(SAMS)。[方法]以酿酒酵母(Saccharomyce cerevisiae)基因组DNA为模板,通过PCR扩增出S-腺苷甲硫氨酸合成酶基因2(sam2),构建重组毕赤酵母(Pichia pastoris)表达载体pPIC9Ks-am2,转化毕赤酵母GS115,以甲醇诱导表达分泌蛋白SAM合成酶,并用HPLC测定重组蛋白的酶活力。[结果]经SDS-PAGE鉴定重组蛋白分子量约50kD,比理论值42.5 kD稍大,可能是分泌过程中糖基化等加工造成。体外酶促反应结果显示,伴随甲醇诱导及初步纯化过程的进行,蛋白活力逐步提高,纯化后比活力为61.48 U/mg。[结论]该研究首次实现了SAM合成酶的胞外表达,为开发和建立体外酶促法生产SAM工艺奠定了基础。

[Objective] The research aimed to study the secreted expression of S-adenosy-L-methionine synthetase（SAMS）in Pichia pastoris.[Method] The gene coding SAMS,from the genomic DNA of Saccharomyces cerevisiae,was amplified by PCR and inserted into the secreted expression vector pPIC9K to get recombinant plasmid.The recombinant plasmid pPIC9K-sam2 was integrated into Pichia pastoris GS115 genome by electroporation and induced by methanol.The activity of the recombinant enzyme was measured using high-performance liquid chromatography（HPLC） by determining the production of the S-adenosyl-l-methionine（SAM） with the enzyme secreted.[Result] The molecular weight of the expression protein identified by SDS-PAGE was about 50 kD,being larger than the theoretical molecular mass of SAMS,which might be due to the glycosylation in the process of secretion.Methanol-induction as well as preliminary purification could enhance the enzyme activity,especially the latter,after which the specific activity of SAMS was improved to 61.48 U/mg.[Conclusion] SAMS with biological activity was secreted successfully in Pichia pastoris GS115 for the first time.And it is the start for the genetic engineering strains to open up prospects for industrial production.