Identification of Mycobacterium tuberculosis signal sequences that direct the export of a leaderless beta-lactamase gene product in Escherichia coli.
Identification of Mycobacterium tuberculosis signal sequences that direct the export of a leaderless beta-lactamase gene product in Escherichia coli.
复制标题
鉴定指导大肠杆菌中无前导序列 β-内酰胺酶基因产物输出的结核分枝杆菌信号序列。
作者：
Anthony J. Chubb;Zenda Woodman;F. M. P. R. D. S. Tatley;H. Hoffmann;R. Scholle;Mario R. W. Ehlers
结核分枝杆菌分泌的蛋白质可能在毒力中起关键作用，也可能构成引发宿主免疫应答的抗原。然而，M.结核蛋白输出机制尚未确定。M的图书馆。构建了连接到信号序列选择载体中的结核病H37 Rv基因组DNA片段，所述信号序列选择载体含有无前导β-内酰胺酶基因和上游Tac启动子。用M.结核病DNA文库中筛选，并在含有50-100微克氨苄青霉素ml-1的平板上筛选，结果在总共14，000个转化体中鉴定出15个Ampr克隆。12个β-内酰胺酶基因融合赋予高水平的Ampr（高达1毫克氨苄青霉素ml-1）;插入片段大小范围从350到3000 bp。在10个完全测序的插入片段中，两个被鉴定为M基因的片段。结核抗原85 A和85 C，它们是该病原体的主要分泌蛋白。其余插入片段中的7个与M.结核病基因组，其中之一编码的蛋白质与来自棒状链霉菌的低亲和力青霉素结合蛋白（PBP）具有35%的同一性。七个假设的ORF中的四个编码具有一个或多个膜相互作用元件的推定输出蛋白，包括脂蛋白附着位点和I型和II型跨膜（TM）片段。所有的插入编码典型的信号序列，除了一个可能的II型膜蛋白。结论：β-内酰胺酶基因融合体在大肠杆菌中的表达是可行的。coli为M.结核病信号序列编码基因。
Proteins secreted by Mycobacterium tuberculosis may play a key role in virulence and may also constitute antigens that elicit the host immune response. However, the M. tuberculosis protein export machinery has not been characterized. A library of M. tuberculosis H37Rv genomic DNA fragments ligated into a signal sequence selection vector that contained a leaderless beta-lactamase gene and an upstream Tac promoter was constructed. Transformation of Escherichia coli with the M. tuberculosis DNA library and selection on plates containing 50-100 micrograms ampicillin ml-1 resulted in the identification of 15 Ampr clones out of a total of 14,000 transformants. Twelve of the beta-lactamase gene fusions conferred high levels of Ampr (up to 1 mg ampicillin ml-1); insert sizes ranged from 350 to 3000 bp. Of ten inserts that were completely sequenced, two were identified as fragments of the genes for M. tuberculosis antigens 85A and 85C, which are the major secreted proteins of this pathogen. Seven of the remaining inserts were > or = 97% identical to hypothetical ORFs in the M. tuberculosis genome, one of which encoded a protein with 35% identity to a low-affinity penicillin-binding protein (PBP) from Streptomyces clavuligerus. Four of the seven hypothetical ORFs encoded putative exported proteins with one or more membrane interaction elements, including lipoprotein attachment sites and type I and II transmembrane (TM) segments. All of the inserts encoded typical signal sequences, with the exception of a possible type II membrane protein. It is concluded that expression of beta-lactamase gene fusions in E. coli provides a useful system for the identification and analysis of M. tuberculosis signal-sequence-encoding genes.