title: [Prokaryotic expression of BirA enzyme and identification of the bioactivity of expressed product].
abstract: OBJECTIVE
  To construct the expression vector of biotin-protein ligase (BirA enzyme) gene and express the BirA enzyme with bioactivity in E.coli BL-21 (DE3).
  
  
  METHODS
  The BirA gene was amplified from E.coli genome by PCR and cloned into pGEX-4T-2 to construct the recombinant plasmid pGEX-BirA. After being verified by DNA sequencing, the fusion protein was expressed under IPTG induction in the E.coli BL-21 (DE3). The expressed product was purified through Glutathione-agarose chromatography column. The enzyme activity of the expressed product was identified by ELISA and Western blot.
  
  
  RESULTS
  The recombinant prokaryotic expression vector pGEX-BirA was constructed and the fusion protein GST-BirA was expressed successfully. The results of ELISA and Western blot showed that the purified BirA enzyme biotinylated HLA-A2 peptide complex.
  
  
  CONCLUSIONS
  BirA enzyme with bioactivity is prepared successfully, which can be used for studying the interaction between protein and protein.
authors.affiliation: Department of Immunology, Tongji Medical College of Huazhong University of Sciences and Technology, Wuhan 430030, China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Li
authors.forename: Xiong-wen
authors.initials: XW
authors.lastname: Wu
authors.forename: Min
authors.initials: M
authors.lastname: Xiong
authors.forename: Xiu-fang
authors.initials: XF
authors.lastname: Weng
authors.forename: Xiao-ling
authors.initials: XL
authors.lastname: Lu
authors.forename: Zhi-hui
authors.initials: ZH
authors.lastname: Liang
authors.forename: Fei-li
authors.initials: FL
authors.lastname: Gong
mesh_terms: D019921:Biotinylation; D015153:Blotting, Western; D019731:Carbon-Nitrogen Ligases / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D015789:HLA-A2 Antigen / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D015789:HLA-A2 Antigen; D012097:Repressor Proteins; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli