大肠杆菌γ-ggt原核表达载体pGEX-4T-1/γ-ggt的构建及其蛋白表达Construction and Characterization of Recombinant Prokaryotic Vector pGEX-4T-1/γ-ggt
胥俊峰;单建华;赵宁伟;殷志敏;
摘要(Abstract):
构建大肠杆菌γ-谷氨酰转肽酶原核表达载体pGEX-4T-1/γ-ggt及表达与鉴定目的蛋白,为进一步利用该酶催化合成茶氨酸等目的产物奠定了基础。以大肠杆菌DH5α总DNA为模板,用PCR法在γ-ggt编码序列上下游引入酶切位点并扩增出γ-谷氨酰转肽酶基因;将γ-ggt编码序列克隆入原核表达载体pGEX-4T-1的相应酶切位点;用PCR鉴定重组质粒pGEX-4T-1/γ-ggt,并对插入基因片断测序;重组质粒pGEX-4T-1/γ-ggt转化大肠杆菌BL21(DE3),经乳糖诱导后用SDS-PAGE分析表达产物,并经Westernblot鉴定。获得大肠杆菌γ-谷氨酰转肽酶基因,并成功构建到原核表达载体pGEX-4T-1上,转化有pGEX-4T-1/γ-ggt工程菌BL21(DE3)经1 g/L乳糖诱导1 h后即开始表达目的蛋白,在诱导6 h后表达蛋白量达到最高,随着时间的延长,目的蛋白没有被降解,此蛋白主要以包涵体形式存在,Western blot鉴定了此目的蛋白。成功构建了大肠杆菌γ-谷氨酰转肽酶原核表达载体pGEX-4T-1/γ-ggt,并进行了目的蛋白的鉴定。
关键词(KeyWords): γ-谷氨酰转肽酶;克隆;表达
基金项目(Foundation): 国家教育部基金项目(2002SWXSBJBC22)
作者(Author): 胥俊峰;单建华;赵宁伟;殷志敏;
Email:
DOI: 10.13989/j.cnki.0517-6611.2007.30.076
参考文献(References):
- [1]KAMATH AB,WANG L,DAS H,et al.Antigens in tea-beverage prime humanVgamma 2Vdelta 2 T cells in vitro and in vivo for memory and nonmemory an-tibacterial cytokine responses[J].Proc Natl Acad Sci USA,2003,100(10):6009-6014.
- [2]TACHIKI T,YAMADA T.-γGlutamyl transfer reaction by glutaminase frompseudomonas nitroreducens IFO12694 and their application for the syntheses oftheanine and-γglutamyl methylamine[J].Biotech Biochem,1998,62(7):1279-1283.
- [3]YAMAMOTO S,WAKAYAMA M,TACHIKI T.Cloning and expression of Pseu-domonas taetrolens Y-30 gene encoding glutamine synthetase:an enzyme avail-able for theanine production by coupled fermentation with energy transfer[J].Biosci Biotechnol Biochem,2006,70(2):500-507.
- [4]SUZUKI H,IZUKA S,MIYAKAWA N,et al.Enzyme production of theanine,anumami component of tea,from glutamine and ethylamine with bacterial-γGlu-tamyl transpeptidase[J].Enzyme and Microbial Technology,2002,31:884-889.
- [5]MEISTER A,ANDERSON M E.Glutathione[J].Annu Rev Biochem,1983,52:711-760.
- [6]TATE S S,MEISTER A.Gamma-glutamyl transpeptidase:catalytic,structuraland function aspects[J].Mol Cell Biochem,1981,39:357-368.
- [7]ALEKSANDR AMISICKA,IWONA MASZCYNSKA,ANDRZEJ W LIPKOWSKI,et al.Synthesis and biological properties of gamma-glutamyl-demorphin,a pro-drug[J].Life Science,1996,58(11):905-911.
- [8]张毅,屈贤铭,杨胜利.乳糖作为诱导剂对重组目的蛋白表达的影响[J].生物工程学报,2000,16(4):464-468.