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 hCE1protein. 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-hCE1monoclonal 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.

The Analysis of SNP within GPR26 Gene and Tumor-Associated Latent Loci

This study aims to investigate the association of SNPs with tumor-associated latent loci. By analyzing the effect of gene mutations on the structure and function of proteins with the database, to speculate the destructive mutants. It was found that individuals with the genotype of the five key SNP loci were more susceptible to tumors; these specific markers can be used as a molecular marker to determine the susceptibility of a tumor to the individual, and to diagnose the population with high risk of tumor; and rs201154887 can be used as a new disease molecular marker.