Construction And Characteristics Of Alkali-tolerance Mutants Of Z Domain For Protein A Chromatography

SpA?Staphylococcus Protein A?can specifically bind to antibody molecules and is widely used in antibodies purification,analysis and testing.Sp A chromatography has become the key technologies for purification of monoclonal antibody and Fc fusion proteins.Traditional SpA is sensitive to alkaline environment and has a poor structure stability,greatly reducing the service life of Sp A chromatography.In this paper,we proposed a new method to improve the stability of the Z domain molecule from Sp A.A couple of alkali-stable mutants of Z domains,Z'and Z",were obtained from several novel mutants,and coupled to Sepharose FF to evaluate the chromatographic performance and the stability in alkaline solution of SpA adsorbents.In this paper,two more stable Z'and Z"domains mutated from Z domain were obtaind.The only difference between the two domains was that the Trp at positio n 12of the mutation Z"domain wa s replaced by the more hydrophobic Ile in the mutation Z'domain.The change trend of the Z,Z'and Z?domain was consistent with the increase of the K yte-Doolittle hydrophobicity index of Trp?-0.9?,Ala?1.8?and Ile?4.5?residues at position 12 in the domains,suggestting that the introduction of more hydrophobic amino acid residues at position 12 of Z domain facilitates increasing the alpha helix content.And the hydrophobic interaction within the Z'domain was enhanced,thereby significantly improving the molecular stability.We further designed and synthesized the tetrameric fusion protein Z₄ and Z'₄,acquired with fermentation expression in Escherichia coli BL21?DE3?.The induction parameters of the recombinant protein A?IPTG concentration,induction time and induction temperature?were optimized.The result showed that the maximum yield was achieved at 0.3 mmol/L IPTG after 3 h of fermentation.And the induction temperature can be selected according to the actual situation.Ion exchange chromatography?Q Sepharose FF?can efficiently purify recombinant SpA with purity up to 95%after the parameters were optimized for the purification of the recombinant SpA.Z SepoFF?Z'SepFF?Z"SepFF?Z₄ SepFF and Z'₄ SepFF were obtained by coupling the novel domains to Sepharose 6 FF matrix.The suitable ligand density was determined,and the static,dynamic adsorption capacity and alkali resistance of the chromatographic medium were determined.Compared to Z SepFF and Z"SepFF,Z'SepFF had better alkali resistance,furturely the Z'₄ SepFF was further improved compared to Z SepFF.This research provided a new idea for the molecular modification of SpA chromatographic ligands.