title: Cloning, expression, identification and bioinformatics analysis of Rv3265c gene from Mycobacterium tuberculosis in Escherichia coli.
abstract: OBJECTIVE
  To clone and express Rv3265c gene of Mycobacterium tuberculosis in Escherichia coli (E. coli) under optimistic conditions, obtain and identify protein expressed, analyze the structure and characteristics of the protein using bioinformatics methods for future applications.
  
  
  METHODS
  Rv3265c gene from Mycobacterium tuberculosis H37Rv was amplified by polymerase chain reaction, and was cloned into the pET-30a vector after purification and recovery. The recombinant plasmid was sequenced and expressed in E. coli BL21(DE3), and then purified and identified by western blotting. The essential physical-chemical properties of the protein were predicated by bioinformatics tools, including subcellular location, secondary structure, domains, antigenic epitopes, etc. Tertiary structure of the protein based on homology modeling was established, while multi-sequence homological alignment and phylogenetic analysis were proformed.
  
  
  RESULTS
  The recombinant protein was obtained in soluble fraction from expression system in E. coli BL21(DE3) carrying pET30- Rv3265c plasmid, and Rv3265c gene was expressed correctly. Bioinformatics analysis showed the protein contained no signal peptide and transmembrane helices, located outside of membrane. Secondary structure analysis revealed it contained α-helix, extended strand and random coil, 46.8%, 14.6%, 38.6%, respectively. Furthermore, it possessed six potential antigenic epitopes, one glycosyl transferase domain. A simple three-dimensional model of this protein was constructed by Swiss-model sever. Both sequences and structures were conservative and especial either in gene or in protein.
  
  
  CONCLUSIONS
  Rv3265c gene might be a desirable molecular target for anti-tuberculosis drug and vaccine. The purified protein from expression will be utilized to study the kinetics of L-rhamnosyltransferase and to develope an enzyme assay for screening vaccine or drug.
authors.affiliation: Agriculture School of Hainan University, Haikou 570228, PR China.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Wu
authors.forename: Peng
authors.initials: P
authors.lastname: Zhou
authors.forename: Shiyun
authors.initials: S
authors.lastname: Qian
authors.forename: Xi
authors.initials: X
authors.lastname: Qin
authors.forename: Zhigang
authors.initials: Z
authors.lastname: Fan
authors.forename: Qiongyao
authors.initials: Q
authors.lastname: Fu
authors.forename: Zhinong
authors.initials: Z
authors.lastname: Zhan
authors.forename: Hua
authors.initials: H
authors.lastname: Pei
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D019295:Computational Biology; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006602:Hexosyltransferases / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011487:Protein Conformation
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D006602:Hexosyltransferases