Expression and refolding of a HLAª²A*0203ª²BSP fusion protein and identification of its tetramers

    JIA Qianª²tao, XU Liª²hui, ZHA Qingª²bing, LI Fengª²yao, HE Xianª²hui*, ZENG Yaoª²ying

    Institute of Tissue Transplantation and Immunology, Institute of Bioengineering, College of Life Science and Technology, Jinan University, Guangzhou 510632, China

    [Abstract] AIM: To optimize expression condition of HLAª²A*0203 heavy chain ectodomain fused with a BirA substrate peptide (BSP) (HLAª²A*0203ª²BSP) for E.coli BL21(DE3) transformant and to prepare a functional HLAª²A*0203 tetramer loaded with an antigenic peptide derived from EBNA3596ª²604 of Epsteinª²Barr virus (EBV). METHODS: The temperature, IPTG concentration and inductive duration of HLAª²A*0203ª²BSP fusion protein expressed for E.coli BL21(DE3) transformant were optimized. SDSª²PAGE and Western blot analyses were employed to detect the expressed fusion protein. The monomer of soluble HLAª²A*0203ª²petide was generated from the fusion protein by in vitro refolding of washed inclusion bodies in the presence of ¦Â2ª²microglobulin (¦Â2m) and HLAª²A*0203 restricted EBV EBNA3596-604 peptide (SVRDRLARL, SVR). Refolded and purified monomer was then biotinylated with BirA. Following the purification of the obtained biotinylated monomer, the tetramer was formed by incubation with streptavidinª²PE at a ratio of 4¡Ã1. Flow cytometry (FCM) analysis was performed to determine its binding activity with specific cytotoxic T lymphocytes (CTL). RESULTS: SDSª²PAGE and Western blot showed that the optimized expression condition was overnight induction at 37¡æ with 0.4mmol/L IPTG. The expressed protein of about 34 kDa in the form of inclusion bodies accumulated up to about 30% of total bacterial protein under the optimized expression condition. The monomer of soluble HLAª²A*0203/SVR was successfully generated and purified. Nonª²reducing SDSª²PAGE analysis showed that the biotinylation was above 85%. HLAª²A*0203/SVR tetramer was constructed by mixing the monomer with streptavidinª²PE at a ratio of 4¡Ã1. FCM analysis indicated that this tetramer could bind specific CTL from HLAª²A2+ donors. CONCLUSION: HLAª²A*0203ª²BSP fusion protein was overexpressed in E.coli under the optimized condition. The tetramers of HLAª²A*0203/SVR were prepared from this fusion protein and it possessed binding activity with specific CTL, which provided a powerful tool for direct visualization and quantification of specific CTL from HLAª²A*0203 donors. ......