Cloning, Prokaryotic Expression, and Purification of the Mincle Receptor Protein
Article
Figures
Metrics
Preview PDF
Reference
Related
Cited by
Materials
Abstract:
In this study, genetic engineering techniques were used to construct the recombinant prokaryotic expression vector pET14b-Mincle, which was then transformed into Escherichia coli host strain BL21 (DE3) to induce the expression of the target protein Mincle. The expression product was purified, verified, and refolded using dialysis. Mincle coding sequence fragments were amplified by reverse transcript polymerase chain reaction (RT-PCR), and cloned into the carrier pMD18-T and the prokaryotic expression vector pET14b which containing His-tag. The constructed vector, verified by restriction endonuclease digestion and DNA sequence comparisons, was then transformed into E. coli host strain BL21 (DE3) and expression was induced using IPTG. A recombinant fusion protein with a molecular weight of approximately 22 kDa was expressed under optimized induction conditions with 1 mmol/L IPTG at 37 ℃ for 5 h. The specificity of the recombinant protein was confirmed using SDS-PAGE, western blots, and ELISA. Mincle was expressed in the host in the form of inclusion bodies, and was purified using a Ni2 + affinity column and refolded by biofilm dialysis. The purified and dialyzed protein was verified by western blotting and ELISA, and its activity was examined with inactivated whole cells of Candida albicans (SC5314). The specific binding with C. albicans showed that the Mincle recombinant fusion protein was biologically active. Thus, this method produced active protein and forms a basis for future studies.
