[Clone and expression of isocitrate lyase gene in Mycobacterium tuberculosis H37Rv].
OBJECTIVE
To construct recombinant plasmid with isocitrate lyase (ICL) gene of Mycobacterium tuberculosis H37Rv for stable and high level expression of ICL in prokaryotic expression system.


METHODS
The recombinant plasmid with ICL gene (pET30 (a)-Rv0467) was constructed by polymerase chain reaction and cloning. The fusion protein was expressed in E. coli host strain BL21 (DE3). Activity of the fusion protein was studied after it was purified with metal chelating chromatography.


RESULTS
We constructed the plasmid which could highly express Mycobacterium tuberculosis H37Rv ICL. The plasmid was highly expressed in E. coli BL21 (DE3), in which the fusion protein accounted for 30% of total protein content. After having been purified by metal chelating chromatography, the purity of the soluble fusion protein was 90%. The fusion protein had activity of ICL.


CONCLUSIONS
Using the prokaryotic expression system, the ICL gene of Mycobacterium tuberculosis H37Rv was successfully cloned and expressed, which build the basis for screening new anti-tuberculosis drugs with ICL as the target point.
Li
Da-wei
DW
National Laboratory for Screening New Microbial Drugs, Institute of Medicinal Biotechnology, CAMS and PUMC, Beijing 100050, China.
Xiao
Chun-ling
CL
Guan
Yan
Y
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D018014:Gene Transfer Techniques; D007522:Isocitrate Lyase / Q000096:biosynthesis / Q000235:genetics*; D009169:Mycobacterium tuberculosis / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000276:immunology; D010957:Plasmids / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics
D011993:Recombinant Fusion Proteins; D007522:Isocitrate Lyase