title: [Molecular cloning, expression, purification and properties of isocitrate lyase gene from Mycobacterium tuberculosis H37Rv].
abstract: OBJECTIVE
  To obtain recombinant protein with enzymatic activities of isocitrate lyase (ICL).
  
  
  METHODS
  The icl gene was amplified by polymerase chain reaction (PCR) from Mycobacterium tuberculosis H(37)Rv strain genomic DNA and cloned into pET28-a(+) vector. The recombinant protein was expressed in E.coli BL21 (DE3). Enzyme activity of the protein was assayed after purifying with Ni-NTA resin.
  
  
  RESULTS
  The recombinant ICL was purified in a highly active state with a specific activity of about 7.657 x 10(2) micromol x mg(-1) x min(-1). The pH curve indicated that recombinant ICL activity was optimal at pH 7.4. The LC/MS spectrometry showed a 50 603.347 molecular mass of recombinant ICL. The CD spectrum showed that the percentages for alpha- helix, beta- sheet, beta- turn, and random coil were 43.8%, 31.9%, 3.4%, and 20.9%, respectively.
  
  
  CONCLUSIONS
  The icl gene of Mycobacterium tuberculosis H(37)Rv was successfully cloned and expressed. The enzymatic properties demonstrated the purified recombinant protein had activities of ICL. This work can facilitate immunologic research and the discovery of novel antimicrobial agents against Mycobacterium tuberculosis.
authors.affiliation: Institute of Genetic, Fudan University, Shanghai 200433, China.
authors.forename: Fang
authors.initials: F
authors.lastname: Hao
authors.forename: Xiu-ying
authors.initials: XY
authors.lastname: Pei
authors.forename: Xue-lian
authors.initials: XL
authors.lastname: Zhang
authors.forename: Shun-bao
authors.initials: SB
authors.lastname: Zhang
authors.forename: Xu-hui
authors.initials: XH
authors.lastname: Lai
authors.forename: Hong-hai
authors.initials: HH
authors.lastname: Wang
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D003001:Cloning, Molecular; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D007522:Isocitrate Lyase / Q000235:genetics*; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D007522:Isocitrate Lyase