Simplification of the purification of heat stable recombinant low molecular weight proteins and peptides from GST-fusion products

The synthesis and purification of peptides of importance in the fields of research and medicine continue to be a challenging task. Chemical synthesis of oligopeptides, especially those greater than 25 amino acids, is cost prohibitive. On the other hand, several bottlenecks exist in the production of recombinant short peptides in heterologous expression hosts such as Escherichia coli (E. coli). In this study, a rapid, cost-effective, and reliable method for the production and single-step-purification of peptides and small proteins was developed. Five peptides and small proteins were overexpressed in E. coli as GST-fusion products in high yields. The recombinant peptides or proteins were successfully purified after enzymatic cleavage with selective heat-induced precipitation of the GST-affinity tag. Qualitative and quantitative analysis using SDS-PAGE and mass spectrometric methods suggest that the recombinant peptides/ proteins were purified to greater than 95% homogeneity. Results of biophysical experiments, including multi-dimensional NMR spec-troscopy, show that the purified proteins/ peptides retain their native conformation. Isothermal titration calo-rimetry studies indicate no significant change in the binding affinity of the heat-treated purified product to their interacting partner(s) compared to the recombinant peptides purified by conventional chromatographic pro-cedures without subjecting to heat treatment. In our opinion, the results reported render the purification of recombinant proteins/ peptides of biomedical relevance using our proposed method easy and reliable.

Automated summary

The study developed a rapid, cost-effective, and reliable method for the production and single-step-purification of peptides and small proteins. Qualitative and quantitative analysis showed that the purified proteins/peptides attained over 95% homogeneity. The proposed method makes the purification of recombinant proteins/peptides easy and reliable for biomedical applications.