The second aconitase (AcnB) of Escherichia coli
The second aconitase (AcnB) of Escherichia coli
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DOI：
10.1099/13500872-142-2-389
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发表时间：
1996-02-01
期刊：
影响因子：
2.8
通讯作者：
Guest, JR
中科院分区：
文献类型：
--
作者：
Bradbury, AJ;Gruer, MJ;Guest, JR
从缺乏ACNA的ACNA：：Kan(R)突变体中部分纯化了大肠杆菌第二乌头酸酶(AcnB)，并通过活性染色和与ACNA抗血清的弱交叉反应鉴定了相应的多肽。AcnB基因定位于2.85min(131.6 kb)，位于染色体上先前归属于两个未知ORF的区域。通过感染Kohara lambda-E的lambda acnB噬菌体，可将乌头酸酶的比活力放大5倍。通过诱导含有从受调控的T7启动子表达acnB编码区的质粒(PGS783)的转化子，可获得高达120倍(50%的可溶性蛋白)的表达。从遗传富集源(JRG3171)获得的AcnB蛋白被纯化到大于或等于98%的同源性，经SDS-PAGE和凝胶过滤分析表明是M(R)100000和105000的单体蛋白。与根据核苷酸序列预测的M(R)93500相比，AcnA与AcnB的序列同源性仅为17%，并且AcnA及其相关蛋白(1-2-3-linker-4)的结构域在AcnB(4-1-2-3)中重排。
The second aconitase (AcnB) of Escherichia coli was partially purified from an acnA::kan(R) mutant lacking AcnA, and the corresponding polypeptide identified by activity staining and weak cross-reactivity with AcnA antiserum. The acnB gene was located at 2.85 min (131.6 kb) in a region of the chromosome previously assigned to two unidentified ORFs. Aconitase specific activities were amplified up to fivefold by infection with lambda acnB phages from the Kohara lambda-E. coli gene library, and up to 120-fold (50% of soluble protein) by inducing transformants containing a plasmid (pGS783) in which the acnB coding region is expressed from a regulated T7 promoter. The AcnB protein was purified to greater than or equal to 98% homogeneity from a genetically enriched source (JRG3171) and shown to be a monomeric protein of M(r) 100000 (SDS-PAGE) and 105000 (gel filtration analysis) compared with M(r) 93 500 predicted from the nucleotide sequence, The sequence identity between AcnA and AcnB is only 17% and the domain organization of AcnA and related proteins (1-2-3-linker-4) is rearranged in AcnB (4-1-2-3).