Surface display of recombinant proteins in lower eukaryotes

摘要

Methods for display of recombinant proteins or protein libraries on the surface of lower eukaryotes such as yeast and filamentous fungi are described. The methods are useful for screening libraries of recombinant proteins in lower eukaryotes to identify particular proteins with desired properties from the array of proteins in the libraries. The methods are particularly useful for constructing and screening antibody libraries in lower eukaryotes.

主权项

1. A method for selecting antibodies or antigen-binding fragments thereof for displayability on a lower eukaryote host cell surface, comprising:
(a) providing a host cell that expresses a capture moiety comprising a cell surface anchoring protein fused to a first binding moiety; (b) transforming the host cell with a nucleic acid encoding (i) a light chain immunoglobulin and (ii) a heavy chain immunoglobulin fused to a second binding moiety that is capable of specifically interacting with the first binding moiety that is fused to the cell surface anchoring protein; wherein there is a stop codon in frame with and between the heavy chain and the second binding moiety; and wherein the host cell expresses antibodies or antigen-binding fragments thereof, that comprise said light chain and said heavy chain immunoglobulin, wherein the heavy chain immunoglobulin is not fused to the second binding moiety and not located at the host cell surface, and wherein the heavy chain immunoglobulin is fused to the second binding moiety and located at the host cell surface; (c) contacting the plurality of host cells with a detection means that specifically binds to host cells that have the antibodies or antigen-binding fragments thereof displayed on the surface of the host cell and does not bind to host cells do not have the antibodies or antigen-binding fragments thereof displayed on the surface of the host cell; (d) isolating the host cells with which the detection means is bound, wherein the presence of the detection means bound to the antibodies or antigen-binding fragments thereof on the surface of the host cells indicates the antibodies or antigen-binding fragments thereof are displayed on the lower eukaryote cell surface; wherein the first binding moiety is GABAB-R2, the second binding moiety is GABAB-R1, the cell surface anchoring protein is GPI protein SED-1 and the host cell is Pichia pastoris; wherein the GABAB-R1 and GABAB-R2 subunits are coiled coil peptides that are capable of the specific pairwise interaction.