大肠杆菌Ⅱ型丙酮酸激酶的表达· 纯化及鉴定
Expression,Purification and Identification of Pyruvate Kinase Ⅱ
赵龙 1周贤轩1
作者信息
- 1. 合肥工业大学生物与食品工程学院,安徽合肥230009
- 折叠
摘要
[目的]获得大肠杆菌的Ⅱ型丙酮酸激酶.[方法]首先用MEGA7软件对不同生物丙酮酸激酶的编码基因进行聚类分析;通过聚合酶链式反应(PCR)扩增了大肠杆菌的Ⅱ型丙酮酸激酶基因pykA,将其克隆到pET28a载体中,构建了重组质粒载体pET28a-pykA并在大肠杆菌BL21中实现了Ⅱ型丙酮酸激酶(PykA)的高效表达;利用镍柱亲和层析系统纯化了PykA蛋白,并进行了高效液相色谱(HPLC)检测.[结果]聚类分析结果表明,大肠杆菌Ⅱ型丙酮酸激酶与其他原核生物的丙酮酸激酶氨基酸序列具有高度同源性.HPLC检测发现Ⅱ型丙酮酸激酶PykA在体外可以高效地将ADP转化为ATP.[结论]该研究获得了Ⅱ型丙酮酸激酶,为ATP的合成以及Ⅱ型丙酮酸激酶为基础的ATP再生系统的研究提供了参考.
Abstract
[Objective] The aim was to obtain the type Ⅱ pyruvate kinase of E.coli.[Method] The phylogenetic analysis using MEGA7 software showed high similarity between the a mino acid sequence of typeⅡpyruvate kinase in Escherichia coli and that of other bacteria.Then,the pykA gene encoding type Ⅱ pyruvate kinase of E.coli was amplified by using Polymerase Chain Reaction (PCR),cloned into the pET28a vector,and overexpressed in the host strain E.coli BL21.The resultant product was purified by nickel affinity chromatography,and detected by high perform-ance liquid chromatography (HPLC).[Result] HPLC analysis revealed that PykA efficiently converted ADP to ATP in vitro.[Conclusion] The progress in expression,purification and activity deter mination of type Ⅱ pyruvate kinase will contribute to ATP production and biosynthesis based on the ATP regeneration system.
关键词
Ⅱ型丙酮酸激酶/蛋白表达/高效液相色谱/大肠杆菌/ATP再生系统Key words
Pyruvate kinaseⅡ/Protein expression/High performance liquid chromatography/Escherichia coli/ATP regeneration system引用本文复制引用
出版年
2017
国家科技期刊平台
NSTL
万方数据