Refolding Of Recombinant Reteplase Expressed As Inclusion Bodies Assisted By Disulfide Oxidoreductases DsbA/DsbC In Vitro

Inclusion body refolding issue is poised to play a major role in the production of recombinant proteins rich in disulfide bonds and remains a bottleneck in the downstream process over the last decade. It is of great practical significance to develop efficient strategies and methods for the refolding of insoluble recombinant proteins containing cysteine in multiple numbers. In this thesis, the refolding of recombinant reteplase was investigated in detail by introducing disulfide oxidoreductase DsbA/DsbC into the refolding system. The study mainly focused on the expression and purification of reteplase and the catalytic abilities of DsbA/DsbC during the refolding process.Firstly, the recombinant reteplase plasmid was constructed successfully and over-expressing of reteplase as inclusion bodies in E. coli was obtained, the yield of reteplase IBs was approximately 1.7 g/L culture and the purity was up to 85% under optimal condition. After washed and denatured, refolding of reteplase in vitro was investigated by direct dilution. The key factors were selected under low concentration of denatured protein. Further, protein refolding at high concentration (800μg/mL) was investigated by orthogonal experimental design. The optimized results indicated that the activity yield increased obviously.Secondly, soluble DsbA and DsbC was produced in E. coli and purified by Ni-NTA affinity chromatography. The output of purified DsbA and DsbC was 166 mg per liter broth and 78 mg per liter broth, which purity could reach 98% and 95%, respectively. Then the purified DsbA/DsbC was immobilized onto NHS-activated Sepharose 4 Fast Flow gel with relatively high capacity, and stable performance was achieved. After that, the immobilized DsbA/DsbC was applied to assist the reteplase refolding. The refolding conditions assisted by DsbA/DsbC including the concentration of DsbA/DsbC, redox conditions, temperature, pH and urea concentration were optimized to increase the activity yield. Results showed that DsbC performed more efficiently in assisting reteplase refolding compared with DsbA. The activity yield could reach 65.8%, which was much higher than simple dilution refolding (25.5%).Finally, a conjoint chromatographic column composed of immobilized DsbC and SEC was established to investigate the on-column refolding of reteplase assisted by DsbC. The optimization of operating conditions including SEC volume, elution flow rate and loading sample resulted a further increase in the activity yield well above 81.3%. The conjoint column displayed excellent performance in assisted refolding ability, operation stability and high utilization after recycle of eight consecutive batches.In this thesis, the efficient refolding of reteplase as inclusion bodies was accomplished by introducing DsbA/DsbC into the refolding system to facilitate correct pairing of disulfide bonds, in which DsbC played an important role by catalyzing protein disulfide isomerisation, showing potential application in the refolding of proteins rich in disulfide bonds in vitro.