[In-vitro refolding and biotinylation of soluble HLA-A2-peptide complex]
Xiu-fang Wong1, Hao Chen, Xiong-wen Wu
1Department of Immunology, Tongji Medical College, Huazhong Sciences and Technology University, Wuhan 430030, China.
Aim:
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.
Conclusion:
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.
