Polynucleotide construct capable of displaying fab in a cell-free translation system, and method for manufacturing and screening fab using same

Abstract

The polynucleotide construct of (1) or (2) below is used to perform ribosome display, CIS display and/or mRNA display in order to screen a Fab against an antigen of interest: (1) a polynucleotide construct which monocistronically comprises a ribosome-binding sequence, Fab first chain-coding sequence, linker peptide-coding sequence, Fab second chain-coding sequence and scaffold-coding sequence in this order, and further comprises at its 3′-end a structure necessary for maintaining a complex with the Fab encoded by itself; and (2) a polynucleotide construct which comprises a Fab first chain-expressing cistron and a Fab second chain-expressing cistron each containing a ribosome-binding sequence, a Fab first chain-coding sequence or Fab second chain-coding sequence, and a scaffold-coding sequence in this order, the first Fab-expressing cistron further comprising at its 3′-end a ribosome stall sequence, said Fab second chain-expressing cistron further comprising at its 3′-end a structure necessary for maintaining a complex with the Fab encoded by itself.

1 . A method for maximizing the affinity of a target-substance-binding protein to a target substance, said method comprising the steps of:
 (I) constructing single-position libraries wherein one amino acid among all amino acid positions constituting a target-substance-binding site in a target-substance-binding protein is randomized to all the 20 types of naturally-occurring amino acids, to provide as many single-position libraries as the number of the all amino acid positions;   (II) constructing a primary library by integrating all of, or an appropriate unit of, these single-position libraries;   (III) selecting said primary library using a protein display system based on the affinity to a target;   (IV) determining the polynucleotide sequence information of said selected sample of the primary library;   (V) extracting single amino acid substitutions frequently observed in said nucleotide sequence information;   (VI) constructing a secondary library comprising combinations of said frequently observed single amino acid substitutions; and   (VII) selecting said secondary library using a protein display system based on the affinity to a target.   
     
     
         2 . The method according to  claim 1 , wherein the step of determining the polynucleotide sequence information of said c selected sample of the primary library is carried out using a next-generation sequencer. 
     
     
         3 . The method according to  claim 2 , wherein said target-substance-binding protein is a full-length antibody or an antibody fragment and said target-substance-binding site is a CDR region. 
     
     
         4 . The method according to  claim 3 , wherein said protein display system is ribosome display, CIS display, mRNA display, phage display, bacterial surface display, yeast cell surface display, or cell surface display with a higher eukaryote. 
     
     
         5 . The method according to  claim 2 , wherein said protein display system is ribosome display, CIS display, mRNA display, phage display, bacterial surface display, yeast cell surface display, or cell surface display with a higher eukaryote. 
     
     
         6 . The method according to  claim 1 , wherein said target-substance-binding protein is a full-length antibody or an antibody fragment and said target-substance-binding site is a CDR region. 
     
     
         7 . The method according to  claim 6 , wherein said protein display system is ribosome display, CIS display, mRNA display, phage display, bacterial surface display, yeast cell surface display, or cell surface display with a higher eukaryote. 
     
     
         8 . The method according to  claim 1 , wherein said protein display system is ribosome display, CIS display, mRNA display, phage display, bacterial surface display, yeast cell surface display, or cell surface display with a higher eukaryote.