酿酒酵母2B-39 sam2在大肠杆菌中的克隆及表达

Clonging and Expression of S-adenosylmethionine 2B-39 Synthetase Gene in E. coli

构建S-腺苷甲硫氨酸合成酶基因(sam2)的基因工程菌,为S-腺苷甲硫氨酸(S-adenosylmethionine,SAM)的工业化生产提供参考。应用PCR技术从酿酒酵母的总DNA中扩增出1.2kb的sam2,构建重组载体pET28a-sam2,将其转入大肠杆菌进行表达和分析。SDS-PAGE显示重组克隆表达的SAM2分子量约47kD,重组蛋白量约细菌总蛋白的20.1%,酶活达到19.6nmol/(h.mL),大肠杆菌表达出了具有生物活性的sam2。

Purpose of the study is to construct S-adenosylmethionine synthetase（sam2） genetic strains for the references to industrial production of S-adenosylmethionine （SAM）. 1.2kb Sam2 from Saceharomyces cerevisiae was obtained by using the technology of polymerase chain reaction （PCR）. Sam2 was linked to pET28a, then the new vector pET28a-sam2 was constructed, pET28a-sam2 was transformed into E. coli. Sam2 was expressed in E. coli and analyzed by method of Sodium Sodeeyl Sulphate-Polyacrylamide Gel Electrophoresis （SDS-PAGE）. The result shows that the relative molecular mass of the recombinant protein is about 47KD. The ratio of recombinant protein expressed in E. coli is up to 20. 1% and its activity up to 19. 6nmol/h · mL. The sam2 with biological activity is expressed in E. coli.