초록 ▼

The present invention provides a genetic method of tethering polypeptides to the yeast cell wall in a form accessible for binding to macromolecules. Combining this method with fluorescence-activated cell sorting provides a means of selecting proteins with increased or decreased affinity for another

The present invention provides a genetic method of tethering polypeptides to the yeast cell wall in a form accessible for binding to macromolecules. Combining this method with fluorescence-activated cell sorting provides a means of selecting proteins with increased or decreased affinity for another molecule, altered specificity, or conditional binding. As one embodiment, attaching an scFv antibody fragment to the Aga2p agglutinin effectively mimics the cell surface display of antibodies by B cells in the immune system for affinity maturation in vivo. As another embodiment, T cell receptor mutants can be isolated by this method that are efficiently displayed on the yeast cell surface, providing a means of altering T cell receptor binding affinity and specificity by library screening.

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1. A method of producing a yeast cell displayed variant ligand binding protein with enhanced binding properties relative to a wild-type of said ligand binding protein, the method comprising: isolating a gene encoding said wild-type binding protein;creating a library of mutated proteins by randomly m

1. A method of producing a yeast cell displayed variant ligand binding protein with enhanced binding properties relative to a wild-type of said ligand binding protein, the method comprising: isolating a gene encoding said wild-type binding protein;creating a library of mutated proteins by randomly mutating said wild-type protein;incorporating each said mutated protein into respective expression cassettes, each having the structure5′-GAL 1-10 promoter—a Aga2p—mutated polypeptide;incorporating each said expression cassette into a respective vector;transforming yeast cells with said cassette-containing vectors to yield a multiplicity of transformed yeast cells;expressing said cassettes in said transformed yeast cells, whereby said ligand binding protein is displayed on the surface of each said yeast cell, said displayed ligand binding protein containing one of said mutated binding;labeling the displayed proteins on said yeast cells by binding a specific label to said displayed proteins;employing flow cytometry to sort said yeast cells according to their labeling characteristics;determining the surface expression level of said ligand binding protein in said sorted cells;determining the ligand binding characteristics of said ligand binding protein on the surface of said sorted cells whereby at least one preferred yeast cell expressing an abundance of ligand binding protein which exhibits enhanced ligand binding characteristics is identified; andcloning said at least one preferred yeast cell. 2. The method of claim 1 wherein said protein is joined at its N-terminus to the C-terminus of a first epitope tag sequence, the N-terminus of said first epitope tag sequence joined to C-terminus of an agglutinin subunit Aga2p sequence, the protein sequence being joined at its C-terminus to a second epitope tag, said Aga2p being joined by two disulfide bonds to an agglutinin subunit Aga1p on said yeast cell surface. 3. A method of producing a variant T cell binding protein with enhanced T-cell binding properties relative to a wild-type of said T cell binding protein, the method comprising: isolating a gene encoding said wild-type T cell binding protein;creating a library of mutated proteins by randomly mutating said wild-type protein;incorporating each said mutated protein into respective expression cassettes, each having the structure5′-GAL 1-10 promoter —a Aga2p—HA—mutated polypeptide—c-myc-3′;incorporating each said expression cassette into a respective vector;transforming yeast cells with said cassette-containing vectors to yield a multiplicity of transformed yeast cells;expressing said cassettes in said transformed yeast cells, whereby a unique fusion protein is displayed on the surface of each said yeast cell, said fusion protein containing one of said mutated T cell binding proteins joined at its N-terminus to the C-terminus of a first epitope tag sequence, the N-terminus of said first epitope tag sequence joined to C-terminus of an agglutinin subunit Aga2p sequence, the T cell binding protein sequence being joined at its C-terminus to a second epitope tag, said Aga2p being joined by two disulfide bonds to an agglutinin subunit Aga1p on said yeast cell surface;labeling the fusion proteins on said yeast cells by binding cytometrically distinguishable labels to said c-myc and to said T cell binding protein;employing flow cytometry to sort said yeast cells according to their labeling characteristics;determining the surface expression level of T cell binding protein in said sorted cells; anddetermining the ligand binding characteristics of said T cell binding protein on the surface of said sorted cells whereby at least one preferred yeast cell expressing an abundance of fusion protein which exhibits enhanced T cell binding characteristics is identified;cloning said at least on preferred yeast cell; andreducing said disulfide bonds whereby said fusion protein is released from said yeast cells. 4. A variant T cell product of the method of claim 3. 5. A process of developing a mutant polypeptide exhibiting more favorable binding of a predetermined ligand relative to the binding characteristics of a wild-type of said polypeptide for said ligand, the process comprising: randomly mutating a predetermined wild-type polypeptide to yield a population of mutated polypeptides;creating a library of yeast cells, each of which displays on its surface at least one copy of a fusion protein containing one of said mutated polypeptides, the amino acid sequence of said fusion protein consisting of said mutated polypeptide sequence joined at its N-terminus to the C-terminus of an agglutinin subunit Aga2p sequence, said Aga2p being joined by two disulfide bonds to an agglutinin subunit Aga1p on said yeast cell surface, a first epitope tag sequence between said Aga2p and ligand binding polypeptide sequences, and a second epitope tag sequence joined to the C-terminus of said ligand binding polypeptide sequence, wherein a label is bound to at least one of said second epitope tag and said mutant polypeptide;sorting said yeast cells by flow cytometry;cloning cells expressing a desired mutant polypeptide;rescuing and sequencing the DNA sequence coding for said desired mutant polypeptide;amplifying and expressing said DNA sequence; andharvesting the desired mutant polypeptide. 6. The DNA sequence of the process of claim 5, said DNA sequence coding for the desired mutant polypeptide product of said process. 7. In a process for developing a protein with enhanced binding characteristics against a predetermined ligand relative to the binding characteristics of a wild-type of said protein for said ligand, in which the process includes mutating a gene encoding a wild-type of said protein, displaying on a yeast cell surface a mutant protein encoded by said mutant gene, contacting said ligand with said displayed mutant protein, and determining the extent of binding of ligand by said displayed mutant protein, the improvement comprising: displaying on said yeast cell surface a fusion protein consisting of a mutant polypeptide sequence joined at its N-terminus to the C-terminus of a first epitope tag sequence, the N-terminus of said first epitope tag sequence joined to the C-terminus of an agglutinin subunit Aga2p sequence, the mutant polypeptide sequence being joined at its C-terminus to a second epitope tag, said Aga2p being joined by two disulfide bonds to an agglutinin subunit Aga1p on said yeast cell surface;labeling said fusion protein by binding a distinctive label to at least one of said second epitope tag and said mutant polypeptide;employing flow cytometry to sort yeast cells according to their labeling characteristics;determining the surface expression level of said fusion protein in said sorted cells; anddetermining the ligand binding characteristics of said mutant polypeptide. 8. A kit for producing a yeast cell displayed variant ligand binding protein with enhanced binding properties relative to a wild-type of said ligand binding protein, the kit comprising: expression cassettes capable of being covalently ligated to individual members of a library of randomly mutated genes encoding a mutated polypeptide, said cassettes each having the structure 5′-GAL 1-10 promoter—a AGA2p—mutated polypeptide;a vector capable of accepting said expression cassettes;said vector useable with yeast cells to yield a multiplicity of transformed yeast cells, capable of expressing said cassettes in said transformed yeast cells, whereby said ligand binding protein is displayed on the surface of each said yeast cell, said displayed ligand binding protein containing one of said mutated binding proteins;labels for labeling the displayed proteins on said yeast cells by binding a specific label to said displayed proteins, said labels being readable by flow cytometry when used to sort said yeast cells according to their labeling characteristics;instructions for determining the surface expression level of said ligand binding protein in said sorted cells, and for determining the ligand binding characteristics of said ligand binding protein on the surface of said sorted cells whereby at least one preferred yeast cell expressing an abundance of ligand binding proteins which exhibits enhanced ligand binding characteristics is identified, and for cloning said at least one preferred yeast cell. 9. A kit for producing a variant T cell binding protein with enhanced T-cell binding properties relative to a wild-type of said T cell binding protein, the kit comprising: expression cassettes capable of being covalently ligated to a gene encoding said wild-type T cell binding protein, each having the structure 5′-GAL 1-10 promoter—a AGA2p BHA—mutated polypeptide Bc-myc-3′;a vector capable of accepting said expression cassettes;said vector useable with yeast cells to yield a multiplicity of transformed yeast cells, capable of expressing said cassettes in said transformed yeast cells, whereby said mutated T cell binding proteins are joined at the N-terminus to the C-terminus of a first epitope tag sequence, the N-terminus of said first epitope tag sequence joined to C-terminus of an agglutinin subunit Aga2p sequence, the T cell binding protein sequence being joined at its C-terminus to a second epitope tag, said Aga2p being joined by at least one disulfide bond to an agglutinin subunit Aga1p on said yeast cell surface;labels for labeling the displayed proteins on said yeast cells by binding a specific label to said displayed proteins, said labels being readable by flow cytometry when used to sort said yeast cells according to their labeling characteristics, said labels cytometrically distinguishable when used to label c-myc and to said T cell binding protein;instructions for determining the surface expression level of said T cell binding protein in said sorted cells, and for determining the ligand binding characteristics of said T cell expressing an abundance of T cell binding proteins which exhibits enhanced T cell binding characteristics is identified, and for cloning said at least one preferred yeast cell, and for reducing said at least one disulfide bond whereby said fusion protein is released from said yeast cells.