[Expression and refolding of a HLA-A*0203-BSP fusion protein and identification of its tetramers].

AIM

To optimize expression condition of HLA-A0203 heavy chain ectodomain fused with a BirA substrate peptide (BSP) (HLA-A0203-BSP) for E.coli BL21(DE3) transformant and to prepare a functional HLA-A*0203 tetramer loaded with an antigenic peptide derived from EBNA3(596-604) of Epstein-Barr virus (EBV).

METHODS

The temperature, IPTG concentration and inductive duration of HLA-A0203-BSP fusion protein expressed for E.coli BL21(DE3) transformant were optimized. SDS-PAGE and Western blot analyses were employed to detect the expressed fusion protein. The monomer of soluble HLA-A0203-peptide was generated from the fusion protein by in vitro refolding of washed inclusion bodies in the presence of beta2-microglobulin (beta2m) and HLA-A*0203 restricted EBV EBNA3(596-604) 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 streptavidin-PE at a ratio of 4:1. Flow cytometry (FCM) analysis was performed to determine its binding activity with specific cytotoxic T lymphocytes (CTL).

RESULTS

SDS-PAGE and Western blot showed that the optimized expression condition was overnight induction at 37 degrees C with 0.4 mmol/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 HLA-A0203/SVR was successfully generated and purified. Non-reducing SDS-PAGE analysis showed that the biotinylation was above 85%. HLA-A0203/SVR tetramer was constructed by mixing the monomer with streptavidin-PE at a ratio of 4:1. FCM analysis indicated that this tetramer could bind specific CTL from HLA-A2+ donors.

CONCLUSION

HLA-A0203-BSP fusion protein was overexpressed in E.coli under the optimized condition. The tetramers of HLA-A0203/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 HLA-A*0203 donors.