Prokaryotic expression and purification of CD3ε chain of human T lymphocyte

Objective: To express the CD3ε chain of human T lymphocytes (hCD3ε) in prokaryotic cells and purify the expressed product. Methods: hCD3ε gene was amplified by RT-PCR using the total RNA of healthy human PBMCs as a template and cloned into vector pCR-II. The constructed recombinant plasmid pCR-II-hCD3ε was identified by restriction analysis and sequencing, then directionally cloned into prokaryotic expression vector pGEX-4T-3, and the constructed recombinant plasmid pGEX-4T-3-hCD3ε was transformed to E. coli BL21 (DE3) for expression under induction of IPTG. The expressed product was purified by GST affinity chromatography and identified by Western blot. Results: Both restriction analysis and DNA sequencing proved that hCD3ε gene was successfully cloned into vector pGEX-4T-3 and expressed stably in prokaryotic cells. The relative molecular mass of expressed product was 49 500. After induction with 0.2 mmol/L IPTG at 25°C for 4.5 h, the expression level of target protein reached a peak value of 29.3% of total somatic protein. A portion of 12.8% of total somatic protein was expressed in a soluble form. The expressed fusion protein reached a purity of 86.1% after purification, and was recognized with rabbit anti-human CD3ε antibody and goat anti-GST antibody. Conclusion: GST-hCD3ε fusion protein was successfully expressed in prokaryotic cells and purified.