A yeast phenotypic screening method for isolation of functional antibodies against g-protein coupled receptors

Abstract

The present invention provides a method for identifying a functional antibody or antigen-binding protein or a fragment thereof that is capable of binding to, and stimulating the activity of, a targettransmembrane protein comprising the steps of providing yeast cells transformed with yeast expression vectors encoding a library of antibodies or antigen-binding proteins or fragments thereof, whereinsaid yeast cells express said target transmembrane protein, expressing said library of antibodies or antigen-binding proteins or fragments thereof in the yeast cells, wherein said expressed antibodies or antigen-binding proteins or fragments thereof are secreted into the periplasmic space of said yeast cells, incubating the yeast cells in or on a selective medium or under a restrictive temperature, or a combination thereof, and detecting a predetermined phenotype in the yeast cells, wherein detection or manifestation of the predetermined phenotype is indicative of binding of the functional antibody or antigen-binding protein or a fragment thereof to said target transmembrane protein and stimulation of said target transmembrane protein by the functional antibody or antigen-binding proteinor a fragment thereof. Antibodies or antibody fragments that are agonists of G-protein coupled receptors identified by the method of the invention are also provided.

Description

technical field [0001] The invention belongs to the field of biotechnology. Specifically, the present invention belongs to the field of antibody isolation and identification through yeast phenotypic screening. Background technique [0002] G protein-coupled receptors (GPCRs), also known as seven transmembrane proteins, are a unique family of receptors with similar structures and diverse functions. Members of this large family exist in all eukaryotes from yeast to humans, and all have core structures unique to this family. The core structure consists of seven transmembrane α-helices connected by six protruding interhelical loops, three of which protrude into the extracellular space with the N-terminus, and three interhelical loops with the C-terminus exposed to the cytoplasm . The extracellular and intracellular domains of GPCRs respectively contain the action site of the ligand and the association site of the cell signal transduction system molecules, so that the receptor...

AI Technical Summary

Problems solved by technology

But such mammalian phenotypic screening methods are unlikely to be applicable to GPCRs, because highly efficient screens like blocking cell death, while convenient for growth factor receptors, are unlikely, or at least not so easily, directly Used for most GPCR receptors in mammalian cell systems

[0007] There is currently no efficient method for isolating and identifying functional antibodies (especially activator antibodies) for GPCRs

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