菠菜叶绿体甘油醛-3-磷酸脱氢酶B亚基及其衍生物在大肠杆菌中的双顺反子系统过表达

Two-cistron system overexpression of chloroplast glyceraldehyde-3-phosphate dehydrogenase subunit B and B-derivatives from spinach in Escherichia coli.

作者信息

Tang G L, Wang Y F, Bao J S, Chen H B

机构信息

State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Fenglin Road, 354, Shanghai 200032, People's Republic of China.

出版信息

Protein Expr Purif. 2001 Jun;22(1):31-7. doi: 10.1006/prep.2001.1413.

DOI:10.1006/prep.2001.1413

PMID:11388796

Abstract

A gene coding for the subunit B (GapB) of chloroplast glyceraldehyde-3-phosphate dehydrogenase from spinach and its two derivatives (GapBc) lacking the GapB-specific C-terminal extension have been cloned by RT-PCR. These three genes have been overexpressed with full activity in Escherichia coli when a two-cistron expression system controlled by an inducible promoter P(trc) is used. With a suitable base composition of the first cistron, the expression level of GapB and the derivatives GapBc are expressed up to 15-20% of the total cell protein and around 20 mg of recombinant GapBcs with full activity are purified from 1 liter of cultured bacteria. The specific activity of the two derivatives GapBc (40-60 u/mg) is similar to that of GapA (50-70 u/mg) and lower than that of reported GapBc derivative (E. Baalmann, R. Scheibe, R. Cerff, and W. Martin, 1996, Plant Mol. Biol. 32, 505-513).

摘要

通过逆转录聚合酶链反应（RT-PCR）克隆了编码菠菜叶绿体甘油醛-3-磷酸脱氢酶亚基B（GapB）及其两个缺失GapB特异性C末端延伸的衍生物（GapBc）的基因。当使用由诱导型启动子P（trc）控制的双顺反子表达系统时，这三个基因已在大肠杆菌中以全活性过量表达。通过第一个顺反子合适的碱基组成，GapB及其衍生物GapBc的表达水平可达总细胞蛋白的15%-20%，并且从1升培养细菌中可纯化出约20毫克具有全活性的重组GapBc。两种衍生物GapBc的比活性（40-60 u/mg）与GapA（50-70 u/mg）相似，且低于已报道的GapBc衍生物（E. Baalmann、R. Scheibe、R. Cerff和W. Martin，1996年，《植物分子生物学》32卷，505-513页）。