Efficient Preparation Of Recombinant Protein AG

Recombinant Protein AG（r-PAG）contains the antibody Fc binding domain of Staphylococcus aureus protein A and the antibody Fc binding domain of protein G.Compared with protein G,it has a wider antibody binding range and has important uses in antibody purification,co-immunoprecipitation,and disease treatment.At present,domestic r-PAG has the problems of high production cost and low efficiency,which seriously affects its large-scale production and application.On the basis of optimizing the expression sequence of r-PAG gene,we construct its high-efficiency expression strain,and optimize the fermentation process of the strain expressing r-PAG.In order to pave the way for the efficient and low-cost production of r-PAG,we purifed the recombinantly expressed r-PAG and used the purified r-PAG for the preparation of affinity chromatography media.Major results are listed below:（1）By optimizing the r-PAG gene sequence and synthesizing the r-PAG gene,the r-PAG recombinant expression plasmid pET28a-r-PAG was successfully constructed.The plasmid was transformed into four different host cells（E.coli BL21（DE3）,E.coli BL21（DE3）p Lys S,E.coli Rosetta（DE3）,E.coli Over Express C43（DE3））,and the recombinant strain was found E.coli Rosetta（DE3）-pET-28a-r-PAG has the best target protein expression and better performance.Therefore,this strain was determined to be the optimal expression strain of r-PAG.The molecular weight of the expressed r-PAG is about 57 k Da,which is consistent with the theoretical size,and the expressed r-PAG is basically a soluble protein.（2）In the shake flask fermentation,the conditions for expression of r-PAG by E.coli Rosetta（DE3）-pET-28a-r-PAG were optimized.It was found that under the optimized conditions(temperature 25℃,IPTG concentration 0.1 mmol/L,the induction time was The cell density OD₆₀₀was 0.6 and the induction time was 9 h).The expression level of the cell r-PAG accounted for 39.8%of the total bacterial protein,and each liter of bacterial solution contained 0.2 g of recombinant protein.In the preparation of r-PAG on the fermentor,it was found that compared with the LB medium fermentation culture,the cell growth and protein expression in the composite medium fermentation culture had advantages.The pH-stat method is used to control the feeding method.The pH is 7.2,the culture temperature is 37℃,the rotation speed is200-650 r/min,the dissolved oxygen content is above 10%,and IPTG is added to 0.1 mmol/L after 14 hours of culture begins to induce,the final cell density OD₆₀₀can reach 62,the expression of r-PAG accounts for about 30%of the cell protein,and each liter of fermentation broth contains 6.1 g of r-PAG.（3）Nickel column affinity chromatography was used to purify r-PAG,and imidazole concentration was linearly eluted to optimize purification.When the imidazole concentration reached 200 mmol/L,r-PAG could be eluted.Under this elution condition,The purity of r-PAG can reach 95.8%,Protein recovery was 60.5%.The loading capacity of the media to the ligand during the preparation of the media was investigated.After calculation,1g r-PAG affinity chromatography media can load 1.09 mg of r-PAG with a coupling efficiency of 27.63%.The resulting affinity chromatography media The molar ligand density is 1.93×10^-8mol/g.The medium was further coupled with the purified r-PAG,and the affinity chromatography medium obtained had a ligand density of 1.09 mg/g and a molar ligand density of 1.93×10^-8mol/g.The adsorption performance of the prepared chromatographic medium to mouse IgG,IgM,IgA and other antibodies was investigated through a static adsorption experiment,and it was found that the medium had an adsorption effect only on mouse IgG,and the saturated adsorption amount Q_mwas 1.17×10^-7mol/g,This shows that the medium can effectively purify and detect mouse monoclonal IgG antibodies without interference from IgA and IgM.