The Phage Display Immunoglobulin Combination Of Molecular Single-domain Random Combinatorial Library Of In Vitro Molecular Evolution

Objective: Through comparing the course of molecule evolutional selection in vitro of the phage-displayed random combinatorial library of single domains of Ig-binding molecules leaded by different Ig molecules and the structure and function of the recombinant Ig-binding molecules obtained by the selection, We aimed to explore the relationship between structure and function of bacteria Ig-binding molecules, the mechanism of the interaction between them and Ig molecules, and the rules of their recombinant and evolution in vitro in order to accumulate experiences of reconstruction of functional proteins using the methods of molecular evolution in future.Methods: The molecular directed evolutional affinity selections in vitro of a phage-displayed random combinatorial library of the A, D domains of protein A, the B2 domains of protein G, the B3 domains of protein L linked by the random linking peptide consisted of 0, 1, 2 or 3 amino acids were processed about 3-4 times using individually human IgG, IgM, IgA antibody molecules and IgGFc constructed by gene engineering as panning targets. We selected eight representative displayed sequences having different structure forms which were obtained by the inducements of different Ig molecules and cloned them to the prokaryotic expression vector PET32a(+), then expressed the fusion proteins with His-Tag on the N terminal, which were purified with Ni-NTA resin ,and characterized their Ig-binding activity by Western blot and ELISA. Result: The combinatorial library showed similar change during the process of affinity selections leaded by IgM and IgA and the displayed sequences were evolved into a common and new structure form: PA-A—PL which doesn't exist in bacteria Ig-binding molecules in nature. We have got six different sequences of this form because of the different sequences of linking peptides, but these sequences trended to appear in the evolution libraries of IgM and IgA at the same time. The evolutional selection leaded by IgG and IgGFc were similar, too and the displayed sequences were also evolved into a common and new structure form: PA-A—PG. We also have got six different sequences because of the difference of the linking sequences, but different with IgM and IgA, only one of the six sequence appeared in the libraries of IgG and IgGFc at the same time, and others appeared separately in each library. Eight purified proteins of the representative displayed sequences were analysed and proved to have good Ig-binding activities, which is similar to the results of evolution. We also found that the PA-A3N—PG—PA-A molecules have strong and specific binding abilities with IgGFc molecule. Conclusion: This is the first report on the construction of the phage-displayed library randomly combined by the single domains of three different Ig-binding molecules and the molecular directed evolutional affinity selection in vitro of the library using four different human Ig molecules as targets. Two new structure forms of Ig-binding molecules which don't exist in the natural molecules were obtained. These structures are all composed of single domains coming from different bacteria Ig-binding molecules, which indicated that Ig-binding domains with different characteristic can form binding complementary predominance. Furthermore, our research also indicated that the linking sequences between each domain had important influence on its Ig-binding function.The study provides a new path of research on the structure and function of Ig-binding molecules and also lay a foundation for directed improvement of Ig-binding molecules and acquirement of new Ig-binding molecules with new Ig-binding characteristics.