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OB-FOLD used as a structure for engineering design of new specific binding agents
OB-FOLD used as a structure for engineering design of new specific binding agents
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机译：OB-FOLD用作新特异性结合剂工程设计的结构
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摘要
A method for obtaining a variant of a starting OB-fold protein that binds to a target and that has a nanomolar affinity or better with said target, wherein said target is a protein or a peptide and wherein said affinity is measured by competition surface plasmon resonance, comprising the steps of a) providing a combinatorial library of variants of said starting OB-fold protein in which 5 to 32 residues involved in the binding of said starting OB-fold protein with their native ligand have been randomized, and optionally wherein said variants additionally comprise an insertion of 1 to 15 random amino acid residues in loop 3 and / or an insertion of 1 to 15 random amino acid residues in loop 4 and / or an insertion of 1 to 20 random amino acid residues in loop 1, and optionally in which 1 to 4 residues of said starting OB-fold protein have been deleted, b) expressing said variants of said library po r presentation in ribosome c) exposing said expressed variants obtained in step b. to said immobilized target, wherein said target is a protein or a peptide d) selecting said variants that bind to said immobilized target e) eluting and recovering the mRNA of said selected variants in step d. f) translate in reverse and amplify said recovered mRNA to obtain a sub-library of selected variants of said starting OB-fold protein g) perform steps b again. to f. from zero to three rounds h) express said variants of said sub-library after step g. by presentation in ribosome i) exposing said expressed variants obtained in step h. to said target that is biotinylated j) incubating this composition comprising said expressed variants and said target of interest of step i. k) capture ternary complexes (mRNA-ribosome-binding agent) bound to biotinylated target with magnetic streptavidin coated beads l) wash said beads and isolate mRNA) reverse translate and amplify said recovered mRNA to obtain a sub-library of selected variants of said starting OB-fold protein n) selecting an element of said sub-library of step m., wherein said element encodes a variant of said starting OB-fold protein comprising from 5 to 32 residues mutated at the contact surface of the native ligand of said starting OB-fold protein and that binds to said target, and wherein the affinity of said variant for said target is a nanomolar affinity or better.
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