Method for in vivo selection of ligand-binding proteins
First Claim
1. A method for selecting a gene by artificially conferring on the gene a preferential ability to be replicated, comprising the steps of:
- (a) rendering non-infectious a replicable genetic package (RGP) which is connected to the product of the gene; and
(b) selectively restoring infectivity to the RGP by allowing the product of the gene to associate with an infectivity mediating complex (IMC);
wherein the IMC comprises a target ligand coupled to a substance which enables the RGP to infect a host cell and be replicated.
3 Assignments
0 Petitions
Accused Products
Abstract
The application discloses a method, and kit, which enables the selection of proteins or other oligo- or polypeptides (collectively, ligand binding proteins, LBPs) which bind with high affinity to a target ligand or receptor. The method relies on the display of the LBP of interest on the surface of replicable genetic packages (RGPs) which are modified so as to be non-infective. Infectivity is restored to those RGPs displaying LBPs with high affinity for a target ligand or receptor by an infectivity mediating complex. The infectivity mediating complex comprises the ligand or receptor covalently linked to a polypeptide which brings about interaction between the RGP and a host cell. It is envisaged that the LBP of interest will belong to a genetically diverse collection of similar substances. The method therefore allows selection of substances with high affinity for target, from within a large collection of variants.
-
Citations
27 Claims
-
1. A method for selecting a gene by artificially conferring on the gene a preferential ability to be replicated, comprising the steps of:
-
(a) rendering non-infectious a replicable genetic package (RGP) which is connected to the product of the gene; and (b) selectively restoring infectivity to the RGP by allowing the product of the gene to associate with an infectivity mediating complex (IMC); wherein the IMC comprises a target ligand coupled to a substance which enables the RGP to infect a host cell and be replicated.
-
-
2. A method for selecting genes encoding ligand or receptor binding peptides or proteins (LBPs) comprising the following steps:
-
(a) modifying a replicable genetic package (RGP) to remove the RGP'"'"'s ability to infect a host cell, such modification being so as to allow infectivity to be restored according to (b) through (d) below; (b) inserting into the genome of the RGP a set of DNA sequences encoding a genetically diverse collection of LBPs; (c) expressing the DNA sequence in a recombinant host cell such that the LBP is displayed at the RGP'"'"'s surface; (d) combining a number of non-infectious RGPs according to (a) and (b) above, host cells, and an infectivity mediating complex (IMC) in a medium, wherein the IMC selectively interacts with one or more of the LBPs, and restores to the RGPs displaying the LBPs their ability to infect the host cells; (e) propagating host cells under conditions in which RGPs becoming associated with the IMC via LBPs displayed on their surface acquire a restored ability to infect the host cells and be replicated; (f) isolating from the host cells RGPs carrying the gene or genes encoding the LBPs, and, optionally; (g) using the RGP again in the same process from (c) above, an additional one or more times; (h) isolating the genes from the RGP. - View Dependent Claims (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)
-
-
20. A kit for the selection of genes encoding ligand or receptor binding peptides or proteins (LBPs) comprising:
-
(a) a vector capable of being packaged as an infectious replicable genetic package (RGP), having the following features; (1) a cloning site which enables introduction of a set of DNA sequences encoding a genetically diverse collection of LBPs in such a way that the LBPs are displayed at the surface of the RGP when the vector is packaged; (2) a modification which removes the RGP'"'"'s ability to infect host cells, such modification being so as to allow infectivity to be restored by interaction between an LBP displayed on the surface of the RGP and a substance which can restore the RGP'"'"'s ability to bind to and infect host cells; and
either or both of;(b) a polypeptide which can be used in the preparation of an infectivity mediating complex (IMC) and which comprises a portion of a protein which, when associated with the RGP, can restore its ability to infect a host cell, and which is able to form a covalent attachment of a ligand to which one or more LBPs is able to bind; and
;(c) a vector having the following features; (1) a first stretch of DNA encoding a portion of an infectivity polypeptide which, when associated with the RGP, can restore its ability to infect a host cell, (2) a second stretch of DNA encoding a linker, (3) a third stretch of DNA comprising a cloning site which allows introduction of one or more DNA sequence(s) encoding (a) ligand(s) or receptor(s) to which one or more of the LBPs is able to bind to a greater or lesser extent. - View Dependent Claims (21, 22, 23, 24, 25, 26, 27)
-
Specification