γ-谷氨酰转肽酶在大肠杆菌周质空间的双融合表达

Dual Fusion Expression of γ-Glutamyltranspeptidase in the Periplasmic Space of Escherichia coli

设计引物PCR扩增大肠杆菌Escherichia coli DH5α的γ-谷氨酰转肽酶(γ-glutamyltranspeptidase,GGT)成熟肽的编码基因,利用基因拼接(gene splicing by overlap extension,SOE)技术将其拼接至小分子泛素相关修饰物(the small ubiquitin-related modifier,SUMO)基因的下游,并重组至pET-39b质粒中,重组质粒转化表达宿主E.coli BL21(DE3)。得到的工程菌经异丙基-β-D-硫代半乳糖苷(isopropyl-β-D-thiogalactoside,IPTG)诱导表达,全菌SDS-PAGE分析,得到表观分子量约为97×103的双融合蛋白,DNA测序进一步证实工程菌构建成功。以菌体直接催化L-谷氨酰胺和乙胺合成L-茶氨酸,纸层析法证实该重组酶具有较好的活性,为酶法生产L-茶氨酸奠定基础。

Primers were designed to amplify the mature peptide ofγ-glutamyltranspeptidase（GGT）coding gene fromE.coli DH5αby PCR.The amplified gene was spliced to the downstream of the gene of the small ubiquitin-related modifier（SUMO）and then was inserted into pET-39 bto obtain the recombinant plasmid which was transformed into the expression host E.coli BL21（DE3）.The engineering strain was induced by isopropyl-β-D-thiogalactoside（IPTG）and then analyzed by SDS-PAGE.The analysis result indicated that the dual fusion protein had an apparent molecular mass of 97×103 approximately.Then the engineering strain was further confirmed to be constructed successfully by DNA sequencing.L-theanine was synthesized fromL-glutamine and ethylamine catalyzed by the thallus obtained after fermentation.And the recombinant enzyme was confirmed to have a remarkable activity by paper chromatography,laying the foundation for the enzymic production of L-theanine.