Thermal Stability Analysis Of Cyclic Protein

Most proteins are linear which adopt folded structures conferring their biologic activties. With the development of protein engineering, more and more enzymes are applied in industry. For the purpose of optimal velocity and economy, conditions for industrial applications are usually different from those for enzymatic reactions in vivo so that their denatured proteins can not be supplemented by freshly synthesized ones. Thermostable enzymes would be a better strategy to eliminate such obstacles. Researchers have established different protocols to improve enzymatic stabilities including screening thermastable mutants from randomly-mutated pools, chemical modifications, adding stabilizing agent, changing salt composition and concentration. However, all protocols stated above require a series of trials.M. Trabi and D. J. Craik et al reported that cyclization can improve apparently the thermastability of protein, suggesting that cyclization may be a better choice for enhancing protein stabilities. Based on these observations, we used Sortase A to cyclize the engineered GFP and ApeRNase HII flanked by GGGG and LPXTG. Further characterization confirmed that cyclization mediated by Sortase A is an efficient method and cyclized proteins exhibit were more thermastable without active damage. Compared with those methods used before, cyclization with sortase A is much more simple and mild, and therefore has more benefits for enzymatic application into industry.