Construction And Selection Of Phage Displayed Library Of Recombinant Ig-binding Molecules

In vitro molecular directed evolution is important means to research on the relationship between structure and function of biomacromolecule. It can chage and optimize the properties of biomacromolecule under laboratorial conditions. Phage display and DNA shuffling are important and efficient techniques for directed molecular evolution . Protein A and protein L are two different bacterial suface proteins which have affinity for Ig molecules, but they have special characteristics respectively. In this study, we randomly combined each mono-domain of protein A and mono-domain of protein L to construct the molecular directed evolutional library of the Ig-binding peptides PALn, using phage display technique combined with in vitro molecular directed evolutional selection in order to explore the relationship between structure and function of IgG-binding molecule, and to lay a foundation for directed improvement of IgG-binding molecule as well. So we carried out the following four-part work. 1. Construction of a novel phagemid pCANTABSSA new cloning site of restriction enzyme Sac I was added to phagemid pCANTAB5S and the open reading frame of the clone sites Stu I, Sal I, Kpn I etc. in phagemid pCANTAB5L was corrected by gene operation. Firstly, The recombinant plasmid pCANTAB5X-IFN- -2b was provided as the amplification template, new cloning site Sac I were added to upstream primer , and Sac I, Stu I and Sal I was added to downstream primer for linker IFN- -2b PCR amplification. The PCR product was cloned into pMD18-T. This DNA fragment was cut out by disgestion of restriction enzymes Xba I and Sal I, and inserted into the clone sites in phagemid pCANTAB5L, by digestion of restriction enzyme Sac I, the linear vector fragment was isolated and ligated to construct the the new phage display vector pCANTAB5S. Sequence analysis showed pCANTAB5S had a new cloning site of Sac I which was added between Xba I and Stu I sites in phagemid pCANTAB5L and the open reading frame of the clone site Stu I in phagemid pCANTAB5L was corrected formore effectively displaying the foreign random peptide and function protein .2. Display of Ig-binding peptides PLn molecular library and selection for positivepeptides from this phage displayed libraryA pair of primers containing Sac I sequence were synthesized to amplify B3 Ig-binding domain of protein L. After digestion of restriction enzyme Sac I, the DNA fragments were ligated to PLn molecule with different length by DNA shuffling. The random DNA library was inserted into Sac I site of pCANTAB5S and was expressed on phage surface as fusion proteins. The capacity and liter of the library were calculated as 3.4 107 phage clones and 6.2x1010TU/ml, respectively. After three rounds IgG affinity selection , Ig-binding peptides 2L was obtained, sequence analysis showed 1 of 12 inserts were L sequences , which suggests the a single B-domain of protein L has affinity for Ig; 8 of 12 inserts were 2L sequences , which suggests the twin B-domain of protein L repeats has good Ig-binding activity. It indicates the application of molecular directed evolution technonogy is a feasible method to explore the structure and function of Ig-binding molecules.3. Display of Ig-binding peptides PALn molecular library and selection for positivepeptides from this phage displayed libraryA pair of primers containing Sac I sequence were synthesized to amplify A,B,C,D Ig-binding domain of protein A and B3 Ig-binding domain of protein L. After digestion of restriction enzyme Sac I, the DNA fragments were ligated to PALn molecule with different length by DNA shuffling. The random DNA library was inserted into Sac I site of pCANTAB5S and was expressed on phage surface as fusion proteins. The capacity and liter of the library were calculated as 3.4xl07phage clones and 6.2x1010TU/ml, respectively. After four rounds IgG affinity selection , a variety of Ig-binding peptides was obtained, sequence analysis showed 20 of 36 inserts were the characteristic (MDPL-MDPA)n structure which consisls of mono-domain of protein A...