Structural Optimization And Application Of The IgG-binding Domains Of Streptococcal Protein G

Streptococcal protein G?SPG?,which binding specifically to the Fc fragment of IgG,plays an important role in the isolation and purification of antibodies.In order to obtain a protein G affinity chromatography medium with high affinity for antibodies,in this study,we optimized and reconstructed the immunoglobulin binding domains of protein G.After analyzing and comparing the adsorption properties of protein ligands to IgG,we examined the purification application of chromatography media with high-load to IgG.The main research contents are as follows:?1?Design of concatemer for binding domain of protein G and construction of Escherichia coli expression system.The C3 region in immunoglobulin binding domain of protein G was used as the structural unit to form three,four,five,six tandem repeats of C3fragments,and a hexa-histidine tag and a cysteine were added to the N-terminus and C-terminus of each concatenation to facilitate the subsequent purification and fixation of the recombinant protein.This experiment successfully constructed four genetically engineered bacteria of E.coli BL21-pET21a-G_3C?E.coli BL21-pET21a-G_4C?E.coli BL21-pET21a-G_5C?E.coli BL21-pET21a-G_6C??2?The acquisition of recombinant protein G.Using E.coli BL21-pET21a-G_4C as a reference strain,the fermentation conditions for high protein expression were determined by optimizing the inoculum size,cell density(OD₆₀₀),IPTG induction concentration and induction time.Four high purity recombinant proteins G were purified by Ni-NTA affinity chromatography,and the purification process of 56 mmol·L^-1imidazole to clean the impurities and 150 mmol·L^-1 imidazole eluting the target protein was determined?3?Screening of high-load affinity chromatography media.We optimized the pH,reaction time,reaction temperature,and protein concentration of the coupling system during preparation of the media,and prepared four affinity chromatography media under optimal conditions.After analyzed and compared the adsorption of the medium,it was determined that the recombinant protein SPG_4C with four C3 fragments had the highest adsorption capacity for hIgG,whose saturated adsorption capacity was 66.79 mg?mL^-1 medium.?4?Performance measurement and application research of chromatography media.The Q_10%dynamic binding capacity to hIgG of SPG_4C was 35 mg·ml^-1,which was higher than 27mg·mL^-11 of GE Protein G Sepharose 4 Fast Flow.After 30 simulated applications the adsorption capacity of the self-made chromatography medium was not less than 85%of the original,and the reusability is good.The purification effect of SPG_4C chromatography medium on human serum and mouse ascites is good,the purity of antibody obtained was around 95%,and the recovery rate was not less than 80%,which was equivalent to or superior to the purification effect of GE Protein G Sepharose 4 Fast Flow.