A method for selecting proteins with improved binding to a label comprising: transforming yeast cells with a vector expressing a protein to be tested fused to a yeast cell wall protein; contacting said yeast cells with a first label which binds to said protein to be tested; determining the level of said first label, wherein a higher level of said label indicates the protein to be tested has improved binding to the label than a protein to be tested that exhibits a lower level of said label.
›10.↳ 9The method of claim 9, further including the steps of: labeling said yeast cells with a second label, wherein said vector used to transform said yeast…d2+9
The method of claim 9, further including the steps of: labeling said yeast cells with a second label, wherein said vector used to transform said yeast cells contains means for expressing a polypeptide sequence fused to said protein to be tested to produce a fusion polypeptide and said second label associates with yeast cells expressing said fusion polypeptide and does not associate with yeast cells which do not express said fusion polypeptide; enriching a transformed yeast population by quantitating said second label, wherein an occurrence of said second label is directly proportional to an abundance of said fusion polypeptides expressed on the cell surface; and comparing said quantitation of said first label to said quantitation of said second label to determine relative binding of said first label to said protein to be tested.
›24.↳ 10The method of claim 10, wherein said polypeptide portion of said fusion polypeptide recognized by said second label is an epitope tag.d3+1
The method of claim 10, wherein said polypeptide portion of said fusion polypeptide recognized by said second label is an epitope tag.
›29.↳ 24The method of claim 24, wherein the epitope tag is c-myc or HA.d4
The method of claim 24, wherein the epitope tag is c-myc or HA.
›25.↳ 10The method of claim 10, wherein said first label and said second label are fluorescent labels.d3+2
The method of claim 10, wherein said first label and said second label are fluorescent labels.
›30.↳ 25The method of claim 25, wherein said quantitation and enrichment steps are performed by fluorescent flow cytometry.d4
The method of claim 25, wherein said quantitation and enrichment steps are performed by fluorescent flow cytometry.
›31.↳ 25The method of claim 25, wherein said quantitation and enrichment steps are performed by fluorescence microscopy.d4
The method of claim 25, wherein said quantitation and enrichment steps are performed by fluorescence microscopy.
›26.↳ 10The method of claim 10, wherein mutagenesis is used to a generate a variegated population of mutants of the protein to be tested.d3+3
The method of claim 10, wherein mutagenesis is used to a generate a variegated population of mutants of the protein to be tested.
›32.↳ 26The method of claim 26, wherein mutagenesis is performed by randomly mutating said vector by passage in a mutator bacterial strain which performs erro…d4
The method of claim 26, wherein mutagenesis is performed by randomly mutating said vector by passage in a mutator bacterial strain which performs error-prone DNA replication.
›33.↳ 26The method of claim 26, wherein mutagenesis is performed by site directed mutagenesis of specific amino acid residues of said protein to be tested.d4
The method of claim 26, wherein mutagenesis is performed by site directed mutagenesis of specific amino acid residues of said protein to be tested.
›34.↳ 26The method of claim 26, wherein selection of mutated proteins of interest with improved binding properties involves iterative cycles of said quantitat…d4
The method of claim 26, wherein selection of mutated proteins of interest with improved binding properties involves iterative cycles of said quantitating and labeling steps.
›11.↳ 9The method of claim 9, wherein said yeast cell wall protein becomes covalently attached to the cell by disulfide bonds during expression.d2
The method of claim 9, wherein said yeast cell wall protein becomes covalently attached to the cell by disulfide bonds during expression.
›12.↳ 9The method of claim 9, wherein the yeast cell wall protein is an agglutinin.d2
The method of claim 9, wherein the yeast cell wall protein is an agglutinin.
›13.↳ 9The method of claim 9, wherein the yeast cell wall protein is the binding subunit of an agglutinin.d2
The method of claim 9, wherein the yeast cell wall protein is the binding subunit of an agglutinin.
›14.↳ 9The method of claim 9, wherein the yeast cell wall protein is Aga2p.d2+1
The method of claim 9, wherein the yeast cell wall protein is Aga2p.
›27.↳ 14The method of claim 14, wherein Aga2p expression is controlled by the GAL1-10 promoter.d3
The method of claim 14, wherein Aga2p expression is controlled by the GAL1-10 promoter.
›15.↳ 9The method of claim 9, wherein the protein to be tested is fused by its N terminus to the C terminus of a binding subunit of an agglutinin.d2
The method of claim 9, wherein the protein to be tested is fused by its N terminus to the C terminus of a binding subunit of an agglutinin.
›16.↳ 9The method of claim 9, wherein the protein to be tested is fused by its N terminus to the C terminus of Aga2p.d2
The method of claim 9, wherein the protein to be tested is fused by its N terminus to the C terminus of Aga2p.
›17.↳ 9The method of claim 9, wherein said yeast cell is of a genus selected from the group consisting of Saccharomyces, Pichia, Hansenula, Schizosaccharomyc…d2+1
The method of claim 9, wherein said yeast cell is of a genus selected from the group consisting of Saccharomyces, Pichia, Hansenula, Schizosaccharomyces, Kluyveromyces, Yarrowia, and Candida.
›28.↳ 17The method of claim 17, wherein said yeast cell is of the species Saccharomyces cerevisiae.d3
The method of claim 17, wherein said yeast cell is of the species Saccharomyces cerevisiae.
›18.↳ 9The method of claim 9, wherein said first label is a magnetic particle attached to a ligand for the protein to be tested.d2
The method of claim 9, wherein said first label is a magnetic particle attached to a ligand for the protein to be tested.
›19.↳ 9The method of claim 9, wherein said protein to be tested is an antibody, Fab, Fv, or scFv antibody fragment.d2
The method of claim 9, wherein said protein to be tested is an antibody, Fab, Fv, or scFv antibody fragment.
›20.↳ 9The method of claim 9, wherein said protein to be tested is the ligand binding domain of a cell surface receptor.d2
The method of claim 9, wherein said protein to be tested is the ligand binding domain of a cell surface receptor.
›21.↳ 9The method of claim 9, wherein said protein to be tested is a soluble ligand of a cell surface receptor.d2
The method of claim 9, wherein said protein to be tested is a soluble ligand of a cell surface receptor.
›22.↳ 9A vector for performing the method of claim 9, comprising a gene coding for said cell wall protein fused to a gene for said protein of interest.d2+7
A vector for performing the method of claim 9, comprising a gene coding for said cell wall protein fused to a gene for said protein of interest.
›35.↳ 22The vector of claim 22, further comprising means for expressing an epitope tag polypeptide sequence fused to said protein of interest.d3+1
The vector of claim 22, further comprising means for expressing an epitope tag polypeptide sequence fused to said protein of interest.
›40.↳ 35The vector of claim 35, wherein said epitope tag polypeptide is selected from the group of HA (SEQ ID NO. 1), or c-myc (SEQ ID NO. 2), or a sequence s…d4
The vector of claim 35, wherein said epitope tag polypeptide is selected from the group of HA (SEQ ID NO. 1), or c-myc (SEQ ID NO. 2), or a sequence selected from the group of DTYRYI (SEQ ID NO. 3), TDFYLK (SEQ ID NO. 4), EEEEYMPME (SEQ ID NO. 5), KPPTPPPEPET (SEQ ID NO. 6), HHHHHH (SEQ ID NO. 7), RYIRS (SEQ ID NO. 8), and DYKDDDDK (SEQ ID NO. 9).
›36.↳ 22The vector of claim 22, wherein said cell wall protein is an agglutinin.d3
The vector of claim 22, wherein said cell wall protein is an agglutinin.
›37.↳ 22The vector of claim 22, wherein said cell wall protein is the binding subunit of an agglutinin.d3
The vector of claim 22, wherein said cell wall protein is the binding subunit of an agglutinin.
›38.↳ 22The vector of claim 22, wherein said yeast agglutinin is Aga2p.d3
The vector of claim 22, wherein said yeast agglutinin is Aga2p.
›39.↳ 22The vector of claim 22, wherein a flexible unstructured polypeptide linker is fused between the cell wall protein and the protein of interest.d3+1
The vector of claim 22, wherein a flexible unstructured polypeptide linker is fused between the cell wall protein and the protein of interest.
›41.↳ 39The vector of claim 39, wherein the said flexible polypeptide linker has the sequence GGGGSGGGGSGGGGS (SEQ ID NO. 20).d4
The vector of claim 39, wherein the said flexible polypeptide linker has the sequence GGGGSGGGGSGGGGS (SEQ ID NO. 20).
›23.↳ 9The method of claim 9, wherein expression of the fusion protein is controlled by an inducible promoter.d2+1
The method of claim 9, wherein expression of the fusion protein is controlled by an inducible promoter.
›42.↳ 23The method of claim 23, wherein the inducible promoter is the GAL1-10 promoter.d3
The method of claim 23, wherein the inducible promoter is the GAL1-10 promoter.
