[Prokaryotic expression of BirA enzyme and identification of the bioactivity of expressed product].
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.
Li
Qing
Q
Department of Immunology, Tongji Medical College of Huazhong University of Sciences and Technology, Wuhan 430030, China.
Wu
Xiong-wen
XW
Xiong
Min
M
Weng
Xiu-fang
XF
Lu
Xiao-ling
XL
Liang
Zhi-hui
ZH
Gong
Fei-li
FL
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*
D029968:Escherichia coli Proteins; D015789:HLA-A2 Antigen; D012097:Repressor Proteins; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli