High level expression of HLA-A*0203-BSP fusion protein in Escherichia coli and construction of soluble HLA-A*0203 monomer and tetramer loaded with Epstein-Barr virus peptide.
Major histocompatibility complex (MHC) tetramer technology is critical for characterization of antigen-specific T cells. In the present study we reported the successful generation of HLA-A*0203 tetramer loaded with Epstein-Barr virus EBNA3(596-604) peptide (SVRDRLARL, SVR). Prokaryotic expression vector for the ectodomain of the heavy chain of HLA-A*0203 fused with a BirA substrate peptide (HLA-A*0203-BSP) was constructed and the expression conditions of the fusion protein in Escherichia coli (E. coli) were optimized. The fusion protein was highly expressed in inclusion bodies within E. coli. It was then refolded in the presence of beta2-microglobulin and SVR peptide to form a soluble HLA-A*0203-SVR monomer. After biotinylation with BirA, the monomer was purified by anion-exchange chromatography and its purity was up to 95%. The tetramer was then formulated by mixing the biotinylated monomer with streptavidin-PE at a ratio of 4:1. Flow cytometry showed that this tetramer could specifically react with antigen-specific CD8+ T cells, indicating that it was biologically functional. These results provide a foundation for further characterization of antigen-specific CD8+ T cells from HLA-A*0203 subjects.
Jia
Qiantao
Q
Institute of Tissue Transplantation and Immunology, Institute of Bioengineering, College of Life Science and Technology, Jinan University, 601 Huangpu Dadao West, Guangzhou 510632, China.
Xu
Lihui
L
Zha
Qingbing
Q
Chi
Xiaoyun
X
Li
Fengyao
F
He
Xianhui
X
D019921:Biotinylation; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000276:immunology; D004926:Escherichia coli / Q000378:metabolism*; D005434:Flow Cytometry; D015234:HLA-A Antigens / Q000737:chemistry* / Q000378:metabolism*; D004854:Herpesvirus 4, Human / Q000737:chemistry*; D006801:Humans; D010455:Peptides / Q000378:metabolism*; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D015183:Restriction Mapping; D012995:Solubility; D014764:Viral Proteins / Q000378:metabolism*
D018076:DNA, Complementary; D000939:Epitopes; D015234:HLA-A Antigens; D010455:Peptides; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins