Anilkumar G, Chauhan M M, Ajitkumar P
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560 012, Karnataka, India.
Gene. 1998 Jul 3;214(1-2):7-11. doi: 10.1016/s0378-1119(98)00248-0.
This study was aimed at the molecular cloning and expression of the gene coding for FtsH protease of Mycobacterium tuberculosis H37Rv (virulent). PCR on the genomic DNA of M. tuberculosis H37Ra (non-virulent) using the oligodeoxynucleotide primers, which were designed based on the codon usage pattern of M. tuberculosis and against the nucleotide (nt) sequence corresponding to two conserved domains of the FtsH protein of Escherichia coli, yielded a 363-bp product. The amino-acid sequence, deduced from the nt sequence of the PCR product, revealed the presence of two ATP-binding motifs and the AAA Signature motif (Second Region of Homology) that are characteristic features found conserved in the FtsH molecules from eubacteria, archaebacteria, and eukaryotes. Southern hybridisation of the NheI digest of the cosmid SCY6F7 containing part of the genomic DNA of M. tuberculosis H37Rv using the PCR fragment as the probe identified the full-length ftsH gene in the 7.2-kb fragment. The gene was subcloned into pBS (SK+) vector, and the FtsH product that was expressed in E. coli transformed with the vector was identified as an 85-kDa protein localised in the membrane.
本研究旨在对结核分枝杆菌H37Rv（有毒力）的FtsH蛋白酶编码基因进行分子克隆和表达。使用基于结核分枝杆菌密码子使用模式设计的寡脱氧核苷酸引物，针对与大肠杆菌FtsH蛋白两个保守结构域相对应的核苷酸（nt）序列，对结核分枝杆菌H37Ra（无毒力）的基因组DNA进行PCR，得到了一个363 bp的产物。从PCR产物的nt序列推导的氨基酸序列显示存在两个ATP结合基序和AAA特征基序（第二同源区域），这些是在真细菌、古细菌和真核生物的FtsH分子中保守的特征性特征。以PCR片段为探针，对含有结核分枝杆菌H37Rv部分基因组DNA的黏粒SCY6F7的NheI消化产物进行Southern杂交，在7.2 kb片段中鉴定出全长ftsH基因。该基因被亚克隆到pBS（SK+）载体中，在用该载体转化的大肠杆菌中表达的FtsH产物被鉴定为一种定位于膜上的85 kDa蛋白。
