Generating Targeted Sequence Diversity in Proteins

摘要

Methods of generating sequence diversity in a protein, such as a ligand-binding protein, are provided. The methods comprise targeted introduction of two or more recombination signal sequences (RSSs) into the protein coding sequence and introduction of the modified protein coding sequence into a recombination-competent host cell, specifically a recombination-competent host cell that is capable of expressing at least RAG-1 and RAG-2, thereby allowing for recombination of the protein coding sequence and expression of variant proteins. Also provided are polynucleotides comprising a nucleic acid sequence encoding a target protein, such as a ligand-binding protein, and comprising two or more RSSs, and compositions and host cells comprising same.

主权项

1. A method of generating variants of a target protein comprising the steps of:
(i) providing a polynucleotide comprising a nucleic acid sequence encoding the target protein and comprising a sequence cassette that comprises a first recombination signal sequence (RSS) linked by an intervening nucleotide sequence to a second RSS, the first RSS capable of functional recombination with the second RSS, wherein the first and second RSS are located in a portion of the nucleic acid sequence that encodes a non-conformational region of the target protein, and wherein the intervening nucleotide sequence is 100 base pairs or more in length; (ii) introducing the polynucleotide into a recombination-competent host cell, and (iii) culturing the host cell in vitro under conditions allowing recombination and expression of the nucleic acid sequence, thereby generating variants of the target protein.