[Expression and refolding of a HLA-A*0203-BSP fusion protein and identification of its tetramers].
OBJECTIVE
To optimize expression condition of HLA-A*0203 heavy chain ectodomain fused with a BirA substrate peptide (BSP) (HLA-A*0203-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-A*0203-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-A*0203-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-A*0203/SVR was successfully generated and purified. Non-reducing SDS-PAGE analysis showed that the biotinylation was above 85%. HLA-A*0203/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.


CONCLUSIONS
HLA-A*0203-BSP fusion protein was overexpressed in E.coli under the optimized condition. The tetramers of HLA-A*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 HLA-A*0203 donors.
Jia
Qian-tao
QT
Institute of Tissue Transplantation and Immunology, College of Life Science and Technology, Jinan University, Guangzhou 510632, China.
Xu
Li-hui
LH
Zha
Qing-bing
QB
Li
Feng-yao
FY
He
Xian-hui
XH
Zeng
Yao-ying
YY
D019921:Biotinylation; D015153:Blotting, Western; D019731:Carbon-Nitrogen Ligases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins / Q000378:metabolism*; D006680:HLA Antigens / Q000235:genetics*; D010455:Peptides / Q000235:genetics* / Q000378:metabolism*; D017510:Protein Folding; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism*
D029968:Escherichia coli Proteins; D006680:HLA Antigens; D010455:Peptides; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli