title: Expression and purification of a functionally active recombinant GDP-mannosyltransferase (PimA) from Mycobacterium tuberculosis H37Rv.
abstract: Lipoarabinomannans (LAM), especially mannose-capped LAM, abundant in the cell wall of Mycobacterium tuberculosis (Mtb) exhibit a broad spectrum of immunomodulatory functions and emerge as key virulence factors that may be relevant drug targets. The pimA gene of mycobacteria encodes a alpha-mannosyltransferase involved in the transfer reaction of the very first mannose from GDP-mannose to the carrier lipid phosphatidyl-myo-inositol, a precursor in the synthesis of LAM. PimA has been proposed to play an essential role in the growth of mycobacteria. In this study, the pimA gene from M. tuberculosis H37Rv was cloned into the pET28a vector and the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) strain, allowing the expression of the Mtb PimA in fusion with a histidine-rich peptide on the N-terminal. The Mtb PimA was purified from the supernatant of the lysed cells under native conditions by immobilized metal affinity chromatography. The purity and molecular weight of Mtb PimA were determined by high performance liquid chromatography and matrix-assisted laser desorption ionization time-of-flight. Circular dichroism spectroscopy study on Mtb PimA showed that the protein was folded. The enzyme assays revealed that Mtb PimA showed a requirement for Mg(2+) for the activity and the K(m) and V(max) values of Mtb PimA were estimated at 18 +/- 2 microM and 0.1 +/- 0.05 nmol/min/microg, respectively. This is the first report describing cloning and expression of GDP-mannosyltransferase gene of M. tuberculosis in E. coli.
authors.affiliation: State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, People's Republic of China.
authors.forename: Xiaoling
authors.initials: X
authors.lastname: Gu
authors.forename: Mao
authors.initials: M
authors.lastname: Chen
authors.forename: Qingzhong
authors.initials: Q
authors.lastname: Wang
authors.forename: Min
authors.initials: M
authors.lastname: Zhang
authors.forename: Baolin
authors.initials: B
authors.lastname: Wang
authors.forename: Honghai
authors.initials: H
authors.lastname: Wang
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002413:Cations, Divalent / Q000737:chemistry; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D006155:Guanosine Diphosphate Mannose / Q000737:chemistry; D007700:Kinetics; D008364:Mannosyltransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D001426:Bacterial Proteins; D002413:Cations, Divalent; D011994:Recombinant Proteins; D006155:Guanosine Diphosphate Mannose; C093299:GDP-mannosyltransferase; D008364:Mannosyltransferases