Generation of highly diverse library of expression vectors via homologous recombination in yeast

Abstract

Methods are provided for generating highly diverse libraries of expression vectors encoding fusion proteins such as single-chain antibodies via homologous recombination in yeast. The method comprises: transforming into yeast cells a linearized yeast expression vector having a 5'- and 3'-terminus sequence at the site of linearization and a library of insert nucleotide sequences that are linear and double-stranded; and having homologous recombination occur between the vector and the insert sequence such that the insert sequence is included in the vector in the transformed yeast cells. The insert sequence comprises a first nucleotide sequence encoding a first polypeptide subunit, a second nucleotide sequence encoding a second polypeptide subunit, a linker sequence encoding a linker peptide that links the first and second polypeptide subunits, and a 5'- and 3'-flanking sequence at the ends of the insert sequence which are sufficiently homologous to the 5'- and 3'-terminus sequences of the linearized yeast expression vector, respectively, to enable homologous recombination to occur. The first polypeptide subunit, the second polypeptide subunit, and the linker polypeptide are expressed as a single fusion protein; and the first and second nucleotide sequences each independently varies within the library of expression vectors.

Description

REFERENCES TO PARENT AND CO-PENDING APPLICATIONS[0001] This application claims the priority of U.S. Application entitled "High throughput generation and screening of fully human antibody repertoire in yeast", Attorney Docket No. 25636-701; U.S. Application entitled "Highly diverse library of yeast expression vectors", Attorney Docket No. 25636-703; and U.S. Application entitled "Methods for screening a highly diverse library of proteins in yeast", Attorney Docket No. 25636-704. The above applications are hereby incorporated by reference.[0002] 1. Field of the Invention[0003] This invention relates to compositions, methods and kits for generating libraries of recombinant expression vectors and using these libraries in screening of affinity-binding pairs, and, more particularly, for generating libraries of recombinant human antibodies and screening for their affinity binding with target antigens.[0004] 2. Description of Related Art[0005] Antibodies are a diverse class of molecules. De...

AI Technical Summary

Benefits of technology

[0120] An advantage of the present invention is that the overall process of generating, selecting and optimizing large, diverse libraries of antibodies mimics the process of natural antibody diversification and maturation in a mammal. In the natural process of antibody affinity maturation, the affinity of the antibodies against their antigen(s) is progressively increased with the passage of time after immunization, largely due to the accumulation of point mutations specifically in the coding sequences of both the heavy- and light-chain variable regions.

Problems solved by technology

In addition, bacteria do not readily process, assemble, or express / secrete functional antibodies.

However, the breeding of such strains of transgenic mice and selection of high affinity antibodies can take a long period of time.

Further, the antigen against which the pool of the human antibody is selected has to be recognized by the mouse as a foreign antigen in order to mount immune response; antibodies against a target antigen that does not have immunogenicity in a mouse may not be able selected by using this technology.

In addition, there may be a regulatory issue regarding the use of transgenic animals, such as transgenic goats (developed by Genzyme Transgenics, Framingham, Mass.) and chickens (developed by Geneworks, Inc., Ann Arbor, Mich

.), to produce antibody, as well as safety issues concerning containment of transgenic animals infected with recombinant viral vectors.

However, due to the long growth circles of plants screening for antibody with high binding affinity toward a target antigen may not be efficient and feasible for high throughput screening in plants.

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