Soluble Expression in Escherichia Coli and Purification of Human Carboxylesterase 1

Objective： To achieve an optimized method for soluble expression of human carboxylesterase 1 （hCE-1） in escherichia coil and purification by Ni2＋-NTA agarose affinity chromatography, to get improved protein yield and purity for further development of hepatocellular carcinoma （HCC） diagnosis ELISA kits. Methods： The best antigen epitopes of hCE1 were predicted by comparing secondary structure, flexible regions, hydrophilicity, antigenic index surface probability of residues. Afterwards, pET-42a （＋） with a His-tag and a GST-tag was applied to form recombinant plasmid pET-42a （＋）/hCE1, which facilitated purification when using Ni2＋-NTA agarose affinity chromatography. Protein quality was measured by SDS-PAGE and BCA protein assay. Western-blot identification was also performed to ensure the correct expression of hCE1 protein. Results： The residues from 500 to 567 near C-terminal of hCE1 protein were considered the best epitopes which exhibited high hydrophilicity and high surface probability and relatively flexible secondary structure and low homology compared with hCE2 and hCE3. His-hCE1 500-567 fusion protein was achieved by IPTG-inducted expression with an expected mass of 42 kDa. After purification, the final product was specially identified, which reached over 95%purity and more than 10 mg/L of microbial culture. In Western blot, the purified fusion protein was recognized by anti-hCE1 monoclonal antibody, along with previous sequencing validation, which demonstrated the correct preparation of soluble hCE1 protein. Conclusion： This is an efficacious and affordable strategy to generate fusion hCE1 of high quality in E coli, which facilitates preparation of hCE1 monoclonal antibody and further HCC diagnosis research.