Study On In-vitro Refolding Of Recombinant Human Interferon-γ

The protein drugs prepared by expression of heterogenous gene in E.coli have been one of important methods to produce genetic engineering pharmaceuticals, but the recombinant proteins are accumulated as inclusion bodies in E.coli. How to refold proteins at high concentration with high yield is one of critical techniques of recombinant proteins production. It has been the bottleneck of industrialization of genetic engineering products. Renaturation of recombinant human interferon-gamma (rhIFN-y) inclusion bodies was studied for promoting to solve the problem.Cultivation conditions of the genetic engineering E.coli (pBV220IFN-yDH5a) which expressed recombinant human interferon-y were investigated. The optimum experiment conditions were gained by determining different culture and induction time and optimizing components of culture medium and fermentation conditions. The optimum experiment conditions were genetic engineering E.coli cultured 8h at 30# and induced 6h at 42#, glucose 2g/L, peptone 15g/L, yeast extract 7.5g/L, NaCl 5g/L, Na2HPO4-12H2O 12.6g/L, KH2PO4 3g/L, NH4C1 l.Og/L, Imol/L MgSO4 2mL/L, shaking speed 220r/min, inoculum concentration 1%, medium volume 20%, pH: 6.8. Results showed that rhIFN-y was expressed about 60% of the total cell proteins and 1.6g inclusion bodies per liter culture medium were obtained under optimized conditions.Protein refolding by dilution is the basis of research on other method of renaturation. The crude inclusion bodies were washed before refolding in order to remove impurities affecting on refolding. Triton X-100 could remove contaminants effectively; the purity of washed inclusion bodies was 88%. The rhIFN-y inclusion bodies could be dissolved by 7mol/L guanidine hydrochloride or 8mol/L urea. The activity and specific activity of rhIFN-y was 2.39.105 lU/mL and 2.21 x107 lU/mg respectively under such experiment conditions: 4癈, pH 8.0, urea concentration Imol/L, protein concentration 0.1mg/mL and pulse renaturation.SP Sepharose Fast Flow ion-exchange chromatography with urea gradient was considered as protein refolding system; it could refold rhIFN-y inclusion bodies effectively at high initial protein concentration and integrate the process of refolding and purification. The specific activity of rhIFN-y was 7.5.l05 lU/mg; the protein yield was 54%; the purity of rhIFN-y was above 95% under such experimentAbstractconditions: protein loading 2.5mg, flow rate 0.5mL/min, urea gradient length 3.4CV and final urea concentration 2mol/L. The high purity of rhIFN-y and no aggregates existing were proved by further Superdex 75 SEC after IEC refolding process.The EBA chromatography can process large amounts of proteins; the proteins adsorbed on STREAMLINE SP could be refolded by decreasing urea concentration gradient; it integrated the process of refolding and purification. The specific activity of rhlFN-y was 8.18xl06 lU/mg; the protein yield was 52.7%; the purity of rhIFN-y was above 90% under such experiment conditions: protein loading 23.2mg, degree of expansion 2.5, urea gradient length 6CV and final urea concentration 3mol/L.Minichaperone (sht GroEL191-345) was produced in 3.7L agitated bioreactor; 216.2 mg Minichaperone per liter fermentation broth was obtained after Ni-NTA affinity chromatography and desalting purification. The total yield was 80%; the purity of protein was above 95%. The rhIFN-y inclusion bodies assisted by free and immobilized Minichaperone could be refolded effectively. The activity of rhIFN-y was 6.63.105 lU/mL and 9.67.105 lU/mL respectively; the protein yield was 32.5% and 47.5% respectively; under such experiment conditions: 15#, 4h, protein concentration 100*g/mL.The conformation of refolded rhIFN-y determined by fluorescence spectra was close to native conformation. It indicated that the unfolding protein was successfully refolded.