Tailoring The Binding Properties Of SpA Ig Binding Domains By In Vitro Molecular Evolution

Immunoglobulin?Ig?-binding proteins?IBPs?are proteins produced by bacteria that specifically bind to host antibodies,including Staphylococcus aureus protein A?Sp A?,Streptococcal Protein G?Sp G?and Peptostreptococcus magnus protein L?PpL?,which is one of the important pathogenic factors of bacteria.IBP,as natural antibody binding molecules,have been widely used in the medical and biological fields,including antibody purification,antibody detection,immunosorbent therapy and so on.Newly evolved lg-bingding molecules?NEIBMs?are based on phage display in vitro molecular evolution technology,through recombined and mutated the antibody binding domains in IBPs,and access to new molecular structure and binding characteristics of antibody binding molecular.SpA is a bacterial immunoglobulin?Ig?-binding protein and has fundamental applications in medical and biological sciences associated with IgG.In addition to its high affinity for IgG Fc,SpA has a low affinity for the VH3 regions of IgG,IgM,and IgA Fab,which may complicate its IgG applications.To diminish its VH3 binding potential and preserve the Fc binding potential,the amino acids at positions 29 and 30 in SpA A domain and at positions 36 and 37 in Sp A C domain which are involved in the interaction with VH3 were randomly mutated respectively,and a combinatorial A_29,30,C_36,37 phage library displaying randomly rearranged mutated A and C domains of Sp A was constructed.The NEIBM,A_L29I30-A_V29K30 and A_V29N30-A_K29V30 were generated by in vitro molecular evolution using unbound antigen and antigen binding of human IgG as bait.The scientific research can be divided into the following four parts.Part 1 The use of unbound antigen and antigen binding of human IgG as bait molecules to in vitro molecular evolution of A_29,30,C_36,37 mutant combination phage library and A,C random combination phage libraryA_29,30,C_36,37 mutant combination phage library and A,C random combination phage library have been successfully constructed in the laboratory.The diversity and randomness of the A_29,30,C_36,37 mutant phage library and the storage capacity of the two libraries all meet the requirements of late molecular evolution.A series of repeated processes were used to extract the A_29,30,C_36,37 mutant combination phage library by?adsorption?–?elution?–?amplification?,and the advantages were combined:A_L29I30-A_V29K30 and A_V29N30-A_K29V30.When the conventional screening of unbound antigen hIgG was used as the bait for the third generation?F₃ generation?,SpA was added as a competitive protein for competitive screening,and monoclonal direct sequencing showed the same advantages as conventional screening:A_L29I30-A_V29K30.Using the antigen binding of hIgG molecule to in vitro mutation screening of the non-mutated combinatorial library,the combination A-A was obtained.Part 2 Prokaryotic expression and purification of A-A and its mutants from in vitro molecular evolutionThe recombinant plasmids p ET-32a?+?-A-A,pET-32a?+?-A_L29I30-A_V29K30 and pET-32a?+?-A_V29N30-A_K29V30 were constructed on the prokaryotic expression vector pET-32a?+?.p ET-32a?+?-A-A,p ET-32a?+?-A_L29I30-A_V29K30and pET-32a?+?-A_V29N30-A_K29V30 were transformed into E.coli BL21 competent cells and induced by IPTG.After purification by N i-NTA column affinity chromatography,SDS-PAGE protein electrophoresis qualitative analysis of the target band size of about 32kDa.Part 3 Analysis of antibody binding properties of A-A and its mutant moleculesBased on the ELISA experiment,the protein was labeled with horseradish peroxidase?HRP?,biotin and other means.It was found that A_L29I30-A_V29K30 and A_V29N30-A_K29V30diminished its VH3 binding potentialand preserved the Fc binding potential,compared with the A-A molecule.The results showed that HRP-A_V29N30-A_K29V30 had the strongest binding potential to IgG,and HRP-A-A had the weakest binding potential to IgG.At the same time,OCTET and SPR were used to verify the results of ELISA.Part 4 Applied research of A-A and its mutantsThe HRP-A-A,HRP-A_L29I30-A_V29K30 and HRP-A_V29N30-A_K29V30 were used to detect anti-HIV antibody,anti-EV71 antibody and anti-CB3 antibody by ELISA.The detection rate of HIV-positive serum was highest in A_V29N30-A_K29V30.The sensitivity of HRP-A_V29N30-A_K29V30 was the best in the detection of anti-EV71VP1 antibody and anti-CB3 antibody,HRP-A-A was the worst.The protein A_L29I30-A_V29K30 was concentrated and dialyzed to form a coupling column.Human serum was purified by agarose column affinity chromatography.A_L29I30-A_V29K30 affinity chromatography was used to recover purified IgG in a comparable amount to SpA,but did not recover IgM and IgA,thus proving the advantage of the former application.In this study,the N EIBM A_L29I30-A_V29K30 and A_V29N30-A_K29V30,with preserved IgG binding potential and diminished IgM and IgA binding potential were obtained through phage-based molecular evolution,and it showed substantial application advantages in IgG purification and detection.This study demonstrates a successful example of functional protein engineering via in vitro molecular evolution and provides useful approaches to remold the Ig binding property of SpA for application purposes.