[In-vitro refolding and biotinylation of soluble HLA-A2-peptide complex].
OBJECTIVE
To refold and biotinylate HLA-A2-peptide complex in-vitro.


METHODS
The BirA substrate peptide (BSP) containing H chain of HLA-A2 and beta(2m) were expressed highly as insoluble aggregates in E.coli, and then the two subunits were refolded to form an HLA-A2-peptide complex by dilution method in the presence of an antigenic peptide (NH(2)-CLGGLLTMV-COOH of EB virus latent membrane protein 2A LMP2A). Then the BirA enzyme was used to biotinylate the refolded complex. The refolded and biotinylated products were detected by ELISA and Western blot with mAb W6/32 and rabbit anti-human beta(2m) antibody and streptavidin.


RESULTS
The refolded complex was composed of H chain aggregate, HLA-A2-peptide complex and beta(2m). Both HLA-A2-peptide complex and the H chain aggregate could be biotinylated.


CONCLUSIONS
The refolding and biotinylation of HLA-A2-peptide complex were successfully performed and the products were confirmed by our practical immunological method. This study laid the foundation for the preparation of HLA-peptide tetramer and artificial antigen presenting cells.
Wong
Xiu-fang
XF
Department of Immunology, Tongji Medical College, Huazhong Sciences and Technology University, Wuhan 430030, China.
Chen
Hao
H
Wu
Xiong-wen
XW
Liang
Zhi-hui
ZH
Han
Jun-yan
JY
Huan
Ya-fei
YF
Gong
Fei-li
FL
D019921:Biotinylation; D019731:Carbon-Nitrogen Ligases / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism; D015789:HLA-A2 Antigen / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D012097:Repressor Proteins / Q000378:metabolism; D012995:Solubility; D014157:Transcription Factors / Q000378:metabolism; D001613:beta 2-Microglobulin / Q000235:genetics / Q000378:metabolism*
D029968:Escherichia coli Proteins; D015789:HLA-A2 Antigen; D012097:Repressor Proteins; D014157:Transcription Factors; D001613:beta 2-Microglobulin; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli