An Integrated System for Library Construction, Affinity Binder Screening and Expression Thereof

Abstract

A recombinant polynucleotide suitable for use in a display vector is provided. The recombinant polynucleotide includes from 5′ to 3′: a first nucleic acid sequence (or insert) encoding an amino acid sequence to be displayed on a surface; a first pre-selected restriction site; a second nucleic acid sequence encoding a surface peptide capable of being displayed on the surface; and a second pre-selected restriction site. Corresponding display vectors that can be converted into expression vectors in a high-throughput fashion, as well as methods of use thereof, are also provided.

1 . A recombinant polynucleotide comprising from 5′ to 3′:
 a first nucleic acid sequence encoding an amino acid sequence to be displayed on a surface; 
 a first restriction site selected from the group consisting of XbaI, NcoI, SalI and XhoI sites; 
 a second nucleic acid sequence encoding a surface peptide capable of being displayed on said surface; and 
 a second restriction site selected from the group consisting of XbaI, NcoI, SalI and XhoI sites; 
 wherein the first nucleic acid sequence is engineered in-frame with the second nucleic acid sequence; 
 wherein the first and second restriction site, when cleaved by corresponding restriction endonuclease thereto, produce compatible sticky ends. 
 
     
     
         2 . The recombinant polynucleotide of  claim 1 , wherein the first nucleic acid sequence encodes an antibody fragment. 
     
     
         3 . The recombinant polynucleotide of  claim 1 , wherein the first and second restriction site each encode amino acids that do not interfere with binding affinity of the amino acid sequence. 
     
     
         4 . The recombinant polynucleotide of  claim 1 , wherein the first and second restriction site each encode amino acids that do not interfere with display of the surface peptide. 
     
     
         5 . The recombinant polynucleotide of  claim 1 , wherein the first restriction site is XbaI site. 
     
     
         6 . The recombinant polynucleotide of  claim 1 , wherein the second restriction site is XbaI site. 
     
     
         7 . The recombinant polynucleotide of  claim 1 , wherein the second nucleic acid sequence encodes a phage coat protein, a yeast outer wall protein, a bacterial outer membrane protein, a cell surface tether domain, or an adapter, or a truncation or derivative thereof. 
     
     
         8 . The recombinant polynucleotide of  claim 1 , wherein the second nucleic acid sequence is gene III of filamentous phage M13, or a truncation or derivative thereof. 
     
     
         9 . The recombinant polynucleotide of  claim 1 , wherein the second nucleic acid sequence encodes an adapter capable of binding to a binding partner, wherein said binding partner is expressed as a fusion and directly displayed on the surface. 
     
     
         10 . The recombinant polynucleotide of  claim 1 , wherein the surface peptide is for phage display, yeast display, bacterial display or mammalian display, or shuttling display therebetween. 
     
     
         11 . The recombinant polynucleotide of  claim 1 , wherein when expressed, the amino acid sequence and the surface peptide are displayed as a fusion protein on the surface. 
     
     
         12 . A display vector for high-throughput conversion into expression vector, comprising:
 the recombinant polynucleotide of  claim 1 ; and   a fusion tag sequence 5′ to the first restriction site or 3′ to the second restriction site.   
     
     
         13 . The display vector of  claim 12 , wherein when the fusion tag sequence is 3′ to the second restriction site, the fusion tag sequence is engineered such that upon (a) removal of the second nucleic acid sequence by cleaving the first and second restriction site, and (b) religation of the compatible sticky ends produced therefrom, the first nucleic acid sequence is in-frame with the fusion tag sequence. 
     
     
         14 . The display vector of  claim 12  wherein the fusion tag sequence is selected from one or more of: an alkaline phosphatase tag, an AviTag, a cutinase tag, a halotag, a flag tag, a c-myc tag, a histidine tag, a GST tag, a green fluorescent protein tag, an HA tag, an E-tag, a Strep tag, a Strep tag II and a YoI 1/34 tag. 
     
     
         15 . The display vector of  claim 12 , provided in a library of display vectors in which each display vector has a unique first nucleic acid sequence. 
     
     
         16 . A method of converting a display vector to an expression vector, comprising:
 providing the display vector of  claim 12 ;   cleaving the first and second restriction site with corresponding restriction endonuclease thereto, thereby producing said compatible sticky ends; and   religating the compatible sticky ends to produce an expression vector in which the first nucleic acid sequence is in-frame with the fusion tag sequence.   
     
     
         17 . The method of  claim 16 , further comprising cleaving within the second nucleic acid sequence to increase religation efficiency in the religating step. 
     
     
         18 . The method of  claim 16 , further comprising, before the religating step, diluting a product from the cleaving step. 
     
     
         19 . The method of  claim 16 , further comprising, after the religating step, introducing said expression vector into a host, to further characterize the first nucleic acid sequence. 
     
     
         20 . The method of  claim 19 , wherein said further characterization includes sequencing the first nucleic acid sequence and/or expressing the first nucleic acid sequence. 
     
     
         21 . The method of  claim 19 , further comprising converting a plurality of display vectors to a plurality of expression vectors in parallel, and introducing said plurality of expression vectors into a population of hosts, thereby performing said method in a high-throughput fashion. 
     
     
         22 . The method of  claim 21 , wherein a conversion rate from the plurality of display vectors to the plurality of expression vectors is higher than 90% or 95%. 
     
     
         23 . A method of identifying an affinity binder to a target, comprising:
 screening a population of first hosts each containing the display vector of  claim 12 , to obtain a subpopulation of first hosts having binding affinity to a target, wherein each first host displays, on a surface of said first host, the amino acid sequence encoded by a unique first nucleic acid sequence in the display vector, and wherein said subpopulation of first hosts each display an affinity binder to said target;   converting display vectors isolated from said subpopulation of first hosts to expression vectors by cleaving the first and second restriction site to remove the second nucleic acid sequence and religating the compatible sticky ends produced therefrom; and   introducing said expression vectors into a population of second hosts, to further characterize the affinity binder.   
     
     
         24 . The method of  claim 23 , performed in a high-throughput fashion. 
     
     
         25 . The method of  claim 23 , wherein a conversion rate of the converting step is higher than 90% or 95%.