In Vitro Evolutional Selection Of A Combinatorial Phage Library Displaying Randomly-rearranged Various Binding Domains Of SpA And SpG With Four Mouse IgG Subclasses

Bacterial immunoglobulin (Ig)-binding proteins (IBPs), such as protein A ofStaphylococcus aureus (SpA) and protein G of group C and G streptococci (SpG), arethe proteins that were studied most. The molecular weight of SpA is about57KDa,contains five highly homologous Ig binding domains such as E, D, A, B, C. Themolecular weight of SpG is about57KDa, contains three highly homologous Ig bindingdomains such as B1, B2, B3. Both SpA and SpG can be combined between CH2γ andCH3γ of a mammalian IgG-Fc, and the binding is stronger with the IgG-Fc. Because ofthese, the SpA and SpG had been widely used for the preparation, purification anddetection of antibodies; immunoprecipitation and immunoadsorption treatment; and hadbecame an important tool for the research, detection and treatment in many fields. Ourlaboratory had constructed many combinatorial phage library displayingrandomly-rearranged various binding domains of the SpA, SpG and PpL, using differentsubclasses and different specieses of IgG molecules as baits, respectively thus generatedthe new species immunoglobulin binding molecules (Novel evolved Ig-bindingmolecules, NEIBMs) such as AL (PpL the B3domain), A-G2, LD3, LD5, EB-B3andthe DA-B3-B3by the in vitro evolution affinity screening. On the bases of the phagedisplay technology of our lab and to further study the Fc binding properties with theSpA and SpG,the in vitro molecular evolution of the combinatorial phage librarydisplaying randomly-rearranged various binding domains of SpA and SpG wasconducted with four mouse IgG subclasses as the baits, respectively. We respect toobtain NEIBMs that have have special binding advantages with the four mouse IgGsubclasses which provid novel candidate molecules for the study of different subclassesof mouse IgG Fc and the binding properties with the SpA or SpG.The whole study consists of three parts as follows: Part I Evolutional selection of the phage library with the mouse IgG subclassesIn vitro molecular evolution of the combinatorial phage library displayingrandomly-rearranged various binding domains of SpA and SpG was conducted withfour mouse IgG subclasses as the baits, respectively. The control phages and the oneinserted domain phages were disappeared and the library was all the two inserteddomains phages, suggesting that the evolution of the library was finished. The controlphages and the one inserted phages disappeared through several rounds of evolutionalselection cycles of coating, incubating, washing and amplying, demonstrating thesuccessful evolution. Each10monoclonal phages were randomly picked up andsequence analysed from the flat of the5,4,5and5using the mouse IgG1, IgG2a,IgG2b and IgG3as baits respectively.Sequence analysed by the software DNAssist showed that DD, DD, AC and DCwere obtained by the mouse IgG1, IgG2a, IgG2b and IgG3as baits respectively whichhave special binding advantages.Part Ⅱ Phage ELISA to verify the binding activity of the three novelcombinatorial molecules with the mouse IgG subclassesTo verify the evolutional selection, phage ELISA was conducted to test the bindingproperties of selective combinatorial molecules: DD, AC and DC.The results showed that when we use one of the mouse IgG as bait to direct the invitro molecular evolution of the combinatorial phage library displayingrandomly-rearranged various binding domains of SpA and SpG and obtained theNEIBMs, thus the binding activity of this NEIBMs would be stronger than others: weuse mouse IgG1as bait to direct the in vitro molecular evolution of the combinatorialphage library displaying randomly-rearranged various binding domains of SpA and SpGand obtained the DD, thus the binding activity of the DD with the mouse IgG1would bestronger than AC and DC; we use mouse IgG2a as bait to direct the in vitro molecularevolution of the combinatorial phage library displaying randomly-rearranged various binding domains of SpA and SpG and obtained the DD, thus the binding activity of theDD with the mouse IgG2a would be stronger than AC and DC; we use mouse IgG2b asbait to direct the in vitro molecular evolution of the combinatorial phage librarydisplaying randomly-rearranged various binding domains of SpA and SpG and obtainedthe AC, thus the binding activity of the AC with the mouse IgG2b would be strongerthan DDand DC; we use mouse IgG3as bait to direct the in vitro molecular evolution ofthe combinatorial phage library displaying randomly-rearranged various bindingdomains of SpA and SpG and obtained the DC, thus the binding activity of the DC withthe mouse IgG3would be stronger than DD and AC. The results were consistent withour affinity screening.Part Ⅲ Prokaryotic expression, purification and binding analysis of the NEIBMsTo further characterize the binding properties of the three NEIBMs. The encodingDNA fragments of the DD, AC and DC were cloned to the prokaryotic expressionvector pET32a(+) to construct the recombinant vectors that were named as pET32a-DD,pET32a-AC and pET32a-DC. The three expression vectors were transformed into EcoliBL21to perform expression. The SDS-PAGE showed a35Kda expressed protein in thesupernatant of cell lysis after IPTG induction. And then DD, AC and DC were labeledwith horseradish peroxidase (HRP) to produce HRP-DD, HRP-AC and HRP-DC. Themouse IgG subclasses and the human IgG binding activity with HRP-DD, HRP-AC,HRP-DC and the Positive control HRP-SpA were detected by binding ELISA assay.The results showed:(1) the binding activity of the HRP-SpA with the mouse IgGsubclasses and the human IgG was significantly higher than that of HRP-DD, HRP-ACand HRP-DC;(2) the binding activity of the HRP-DD, HRP-AC, HRP-DC andHRP-SpA with the mouse IgG subclasses and the human IgG were consistent and allwere: human IgG> IgG3> IgG2a> IgG2b> IgG1.Summary: 1. In vitro molecular evolution of the combinatorial phage library displayingrandomly-rearranged various binding domains of SpA and SpG was successfullyconducted with four mouse IgG subclasses as the baits, respectively, and threeNEIBMs such as DD, AC and DC were obtained.2. Phage ELISA was conducted to further verify the binding activity of the three novelcombinatorial molecules with the mouse IgG subclasses3. We successfully expressed and purified the three NEIBMs: DD, AC and DC, thenthey were all labeled with horseradish peroxidase(HRP). The binding activity of theHRP-DD, HRP-AC, HRP-DC and positive control HRP-SpA with the mouse IgGsubclasses and the human IgG were consistent that all were: human IgG> IgG3>IgG2a> IgG2b> IgG1.In this study, three novel evolved Immunoglobulin binding molecules D-D、A-Cand D-C are obtained from the in vitro molecular evolution of a combinatorial phagelibrary displaying randomly-rearranged various binding domains, and they have specialbinding advantages with the four mouse IgG subclasses that didn’t exist neither in SpAnor SpG. The three molecules provided novel candidate molecules for the study ofdifferent subclasses of mouse IgG Fc and the binding properties with the SpA or SpG.