Cooperation Of Immobilized Metal Afinity Chromatography And Artificial Chaperone On EGFP Inclusion Bodies Refolding

For the purpose of purification and on-column refolding of inclusion bodies by immobilized metal affinity chromatography (IMAC) at the same time, more recombinant proteins were overexpressed with a 6×his-tag. Due to the increasing of the local protein concentration in the column, some proteins would aggregated as precipitation, so the refolding recovery (including fluorescent recovery and protein recovery) was decreased and the column was blocked sometimes. It is reported that the artificial chaperone system (ACS) can shield the hydrophobic point of proteins and prohibit the aggregation of proteins. In this thesis, a novel on-column refolding technique which was the cooperation of IMAC and ACS was proposaled. We investigated the refolding of the recombinant 6×his-tag enhanced green fluorescent protein (EGFP) overexpressed as inclusion bodies by IMAC-ACS and the refolding recovery of it was compared with that of other refolding techniques.The results indicated that IMAC-ACS refolding technique can provide the best refolding recovery. The dilution refolding technique was only adapted to low protein concentration of 0.05 mg·mL-1 ~ 0.2 mg·mL-1 and the fluorescent recovery is about 30% ~ 40%. The ACS refolding technique performed a litter better than dilution refolding. If the protein concentration was up to 0.6 mg·mL-1, the fluorescent recoveries were only 14% and 25% for dilution and ACS refolding respectively. The refolding recovery was obviously improved by IMAC refolding technique. The fluorescent recovery was 60% and the fluorescent recovery was 80% for refolded protein of 0.6 mg·mL-1. By comparison, the IMAC-ACS refolding would promote the refolding recovery more efficiently. Under the same chromatographic condition with IMAC refolding, the fluorescent recovery was 80% and the protein recovery was 82% by IMAC-ACS refolding.To get a more efficient refolding recovery, we optimized the operation conditions. Under the optimum conditions (molar ratio of protein to CTAB: 1/5; binding time: 20 min; flow rate of refolding buffer: 0.4 mL·min-1; uera concentration in refolding buffer: 1.5 mol·L-1; uera concentration in elute: 2.5 mol·L-1), the fluorescent recovery was 87% and the protein recovery was 91%, meanwhile, the refolded protein concentration can reach 0.8 mg·mL-1 ~ 1.0 mg·mL-1. Of course, the increasing loading sample concentration could result in a low refolding recovery, however, the IMAC-ACS refolding technique can also provide a acceptable recovery. Moreover, the refolded protein was analyze d by size-exclusion chromatography (SEC) and circular dichroism (CD). The SEC results demonstrated that there was almost the mono-EGFP in the refolded proteins from IMAC-ACS refolding technique was, whereas, lots of poly-EGFP from other techniques. By the calculation of CD results, the secondary structure of refolded proteins was almost the same as that of native proteins.