Bradbury Alan J, Gruer Megan J, Rudd Kenneth E, Guest John R
The Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2UH, UK.
National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA.
Microbiology (Reading). 1996 Feb;142 ( Pt 2):389-400. doi: 10.1099/13500872-142-2-389.
The second aconitase (AcnB) of Escherichia coli was partially purified from an acnA::kanR 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 center dot 85 min (131 center dot 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 lambdaacnB 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 > or = 98% homogeneity from a genetically enriched source (JRG3171) and shown to be a monomeric protein of Mr 100 000 (SDS-PAGE) and 105 000 (gel filtration analysis) compared with Mr 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).
从缺乏AcnA的acnA::kanR突变体中部分纯化了大肠杆菌的第二种乌头酸酶（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基因位于染色体上先前指定为两个未鉴定开放阅读框的区域中的2.85分钟（131.6 kb）处。用来自Kohara λ-大肠杆菌基因文库的λacnB噬菌体感染后，乌头酸酶的比活性提高了五倍，通过诱导含有质粒（pGS783）的转化体，其中acnB编码区由受调控的T7启动子表达，比活性提高了120倍（占可溶性蛋白的50%）。从基因富集的来源（JRG3171）中将AcnB蛋白纯化至≥98%的纯度，与根据核苷酸序列预测的93500的分子量相比，经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）显示为分子量100000的单体蛋白，经凝胶过滤分析显示为分子量105000的单体蛋白。AcnA和AcnB之间的序列同一性仅为17%，AcnA和相关蛋白的结构域组织（1-2-3-连接区-4）在AcnB中重排为（4-1-2-3）。
