Effect Of Escherichia Coli DsbC On Phytase Refolding In Vitro

Both in the eukaryotes and prokaryotes, correct disulfide bond formation of secreted proteins is very important. Correct disulfide bonds can stabilize protein structure, on the contrary, incorrect disulfide bond formation of proteins may leads to protein aggregation or degradation. DsbC is a disulfide isomerase, which could catalyze the formation of disulfide and isomerization of abnormal disulfide in vivo. So what roles of DsbC plays in refolding of the protein with many disulfide bonds? In this paper, the gene of DsbC was fristly cloned, then the DsbC protein was expressed, purified and renaturation of other proteins. The effect of DsbC and other folding aids on refolding of chemically denatured phytase with five disulfide bonds was investigated. The following results were achieved:1. The gene encoding DsbC was expressed in Escherichia coli with prokaryotic expressive plasmid, pET-30a. After denaturation, the recombinant DsbC was successfully renatured and confirmed to be enzymatically active.2. DsbC facilitated the proper refolding of phytase. When the phytase that was denatured by urea was renatured without DsbC, about 45% of the native enzyme activity was recovered. However, the activity may reach 75% when the phytase was renatured by DsbC. When the enzyme was denatured by urea and DTT, less activity was recovered, only about 7%. However, about 35% of the native enzyme activity was recovered by DsbC. The results indicated that DsbC played an important role in the catalytic activity.3. The result of DsbC facilitating the refolding of phytase showed that DsbC may be both a disulfide isomerase and a molecular chaperone. To tell the difference of functions of DsbC, the thiol group of DsbC was modified by iodoacetamide, which may make DsbC without the functions of disulfide isomerase. When the denatured phytase was renatured by placed into refolding buffer in the presence of DsbC and modified DsbC, about 30% or 9% of the native enzyme activity was recovered. The results indicated that DsbC was both a disulfide isomerase and a molecular chaperone in refolding of phyase, moreover, the activity of disulfide isomerase was the major function.4. The effect of DsbC is related to redox environment. Different ratio of GSH/GSSG affected the recovering of denatured phytase. Osmolytes are a series of small molecules that can maintain the correct conformation of protein by acting as molecular chaperons. Osmolytes play different roles in refolding of denatured phytase. The function of osmolytes was different from different concentrations and different types. All of glycerol, DMSO and proline could facilitate the refolding of phytase. When the denatured phytase was put into refolding buffer containing both DsbC and osmolytes, the recovered activity was further improved. The results indicated that DsbC and osmolytes demonstrated a synergistic effect on protein refolding.