Zhang Min, Wang Jia-dong, Li Zuo-feng, Xie Jun, Yang Yan-ping, Zhong Yang, Wang Hong-hai
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai, PR China.
Protein Expr Purif. 2005 Jul;42(1):59-66. doi: 10.1016/j.pep.2005.03.022. Epub 2005 Apr 12.
Rv3487c (lipF), a member of the lipase family of Mycobacterium tuberculosis, is related to virulence of this pathogen. Real-time RT-PCR analysis indicated that Rv3487c was induced at low pH in M. tuberculosis cultured in vitro. The gene of Rv3487c was cloned and expressed as fusion protein in Escherichia coli. After removal of the N-terminal domain of the fusion partner by enterokinase treatment, the effect of pH, temperature, and detergents on the purified enzyme activity and stability was characterized. Rv3487c could efficiently hydrolyze short chain esters. The catalytic triad of Rv3487c consists of residues Ser90, Glu189, and His219 as demonstrated by amino acid sequence alignment, three-dimensional modeling, and site-directed mutagenesis.
Rv3487c（lipF）是结核分枝杆菌脂肪酶家族的一员，与该病原体的毒力相关。实时逆转录聚合酶链反应分析表明，在体外培养的结核分枝杆菌中，低pH条件可诱导Rv3487c表达。Rv3487c基因被克隆，并在大肠杆菌中表达为融合蛋白。经肠激酶处理去除融合伴侣的N端结构域后，对pH、温度和去污剂对纯化酶活性和稳定性的影响进行了表征。Rv3487c能够高效水解短链酯。通过氨基酸序列比对、三维建模和定点诱变证明，Rv3487c的催化三联体由Ser90、Glu189和His219残基组成。
