玉米谷氨酰胺转胺酶基因的克隆及原核表达

Cloning and Expression of Maize Transglutaminase in Escherichia coli

从玉米嫩叶中提取总RNA,通过RT-PCR的方法扩增出玉米谷氨酰胺转胺酶(TGase)全长基因,回收目的片段并测序,该基因编码区全长1605bp,编码535个氨基酸残基,分子量为60.9kD,与GenBank(登录号:AJ421525)上已发表的序列同源性为100%。按正确的阅读框架将玉米TGase基因片段定向克隆到表达载体pET-28a上,将重组质粒转化到大肠杆菌Rosetta(DE3)菌株,1mmol/L IPTG诱导融合蛋白表达,经凝胶分析软件测得蛋白表达量约占总蛋白的15%,以His-Tag抗体作为一抗,采用Western-blot方法检测目的蛋白,结果证明所表达的特异蛋白是带有His-Tag的重组融合蛋白,利用Ni2+-NTA琼脂糖树脂亲和层析柱纯化目的蛋白,SDS-PAGE鉴定为单一条带,测得纯化后的TGase酶活力达到16U/mg。

In this study,total RNA was extracted from young leaves of maize.Reverse transcription PCR（RT-PCR） method was used to obtain full-length transglutaminase（TGase） gene.The amplified fragment was sequenced to have 1605 bp.The gene consisted of 535 amino acid residues with a calculated molecular mass of 60.9 kD.It was identical to the published TGase gene（GenBank NO.AJ421525）.This gene fragment was cloned into pET-28a expression vector.The pET-28a-TGase was transformed into E.coli Rosetta（DE3） and expressed in E.coli cells with the induction of 1 mmol/L IPTG.According to quantitative analysis,the expression amount of this protein was approximately 15% of total protein.Western-blot analysis confirmed that this protein was a His-tag recombinant protein.After the purification by Ni2＋-NTA affinity chromatography,the purified protein exhibited high purity and one single band was shown in SDS-PAGE.The TGase activity was up to 16 U/mg after purification.