Soluble Expression And Characterization Of Bovine Prochymosin And Snake Venom Thrombin-like Enzyme

In the fields of protein engineering, it is very important to obtain a high expression level of a functional protein in a highly active form. As every protein is different, there can be no single strategy for obtaining different recombinant proteins with high activities. Thus, different strategies must be worked out for different proteins of interest in order to obtain high expression levels of functional proteins.In this work, two representative functional proteins, bovine chymosin and snake venom thrombin-like enzyme were chosen to investigate the strategies for obtaining high expression and activity for a functional protein, since it was difficult to express these two proteins to a high level and with high activity.1. Constitutive expression, purification and characterization of bovine prochymosin in Pichia pastoris GS115Chymosin can specifically break down the Phe105-Met106peptide bond of milk^-casein to form insoluble para-κ-casein, resulting in milk coagulation, a process that is used in making cheese. In this study, in order to obtain an alternative milk coagulant which is safe and efficient, and simultaneously can produce cheese with a good taste, bovine prochymosin B was chosen and constitutively expressed to a high level in P. pastoris. The recombinant chymosin was expressed mainly as a secretory form, and it exhibited milk-clotting activity. It was purified by ammonium sulfate fractionation, anion exchange, followed by cation exchange chromatography. A final yield of24.2%was obtained for the purified enzyme, which appeared as a single band in SDS-PAGE having a molecular mass of approximate36kDa. Proteolysis assay showed that it specifically hydrolyzed κ-casein and heptapeptide substrate. It was stable at25-50℃and had optimal activity at37℃and pH4.0. The activity of the recombinant chymosin was activated by cations such as Mn2+, Fe3+, Mg2+and Na+, but inhibited by K+, Co2+, Zn2+, Ni2+, and to a lesser extent by Cu2+. Digestion by PNGase F and analysis on NetCGlyc1.0Server suggested that recombinant chymosin had not N-glycosidaton. These results suggested that recombinant bovine chymosin is an acid milk coagulant, and it could be considered as a safe and efficient enzyme suitable for use in cheese production. 2. The soluble expression strategy of snake venom thrombin-like enzymeIn China, snake venom thrombin-like enzyme is a basic medicine for the clinical therapy of occlusive thrombi. However, due to the limited resource of snake venom, production of these enzymes from snake venoms faces a big challenge. Therefore, genetic engineering is one of the best alternatives to solve this problem. Currently, high-scale production of this enzyme is hard to accomplish since the expression and activity of thrombin-like enzyme is generallly low. This study explores the expression strategies of gussurobin, a snake venom thrombin-liken enzyme from Agkistrodon halys ussuriensis Emelianov.1) Escherichia coli expression system①Gussurobin with or without three additional amino acids (Cys-Arg-Gly) at its N-termius was expressed in the host cell of E.coli ER2566and E.coli Rossetta-gamiTMB(DE3)plysS using the inducible self-cleaving intein tag as a fusion partner. When using E.coli ER2566as host cell, recombinant protein with Cys-Arg-Gly at its N-terminus was expressed solubly, however, this recombinant enzyme had no fibrinogenlytic activity. The recombinant enzyme without additional amino acids at the N-terminus was expressed as inclusion body in E.coli ER2566host cell. In case of E.coli Rossetta-gamiTMB(DE3)plysS, we couldn’t find the expression product.②Gussurobin was fused with maltose binding protein (MBP) and co-expressed with Dsbc, a disulfide isomerase assisting the folding of proteins, in E.coli ER2566and E.coli Rossetta-gamiTMB(DE3)plysS. When using E.coli ER2566as the host cell, the recombinant gussurobin was soluble, however, this enzyme had no activity. In the case of E.coli Rossetta-gamiTMB(DE3)plysS, we couldn’t find the expression product.2) Pichia pastoris expression systemGussurobin was expressed with MBP fusion tag in P.pastoris which is a eukaryotic expression system. As a result, the expression product was not found and this recombinant enzyme had not fibrinogenlytic activity.