Secreted Expression of S-adenosy-L-methionine Synthetase in Pichia pastoris

WANG Lian-zhe, ZHANG Xian-qing, LI Yang, YANG Guang-xiao, HE Guang-yuan

Objective:The research aimed to study the secreted expression of S-adenosyl-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 S-adenosy-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.

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