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US 6,686,168 B1Granted
Cell surface display of proteins by recombinant host cells
AssigneeZymoGenetics, Inc.
InventorSi Lok
Life of this patent
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Abstract
Methods and vectors are described for expressing recombinant proteins on the surface of host cells. These processes and compositions provide the basis for strategies to produce a fusion protein, comprising a membrane anchor that allows extracellular attachment of the fusion protein in a type II orientation.
Claims36
A method of selecting nucleic acid molecules that encode a member of a complementary/anti-complementary binding pair, comprising:
- (a) transfecting an expression vector into eukaryotic host cells to produce recombinant host cells, wherein the expression vector comprises:
- (i) a eukaryotic promoter,
- (ii) a nucleotide sequence encoding a type II signal anchor domain segment, and
- (iii) a gene, or gene fragment, that encodes the first member of a complementary/anti-complementary binding pair,
- wherein the expression vector comprises elements (i) to (iii) in a 5′ to 3′ order, wherein the gene, or gene fragment, resides in-frame with the nucleotide sequence that encodes the type II signal anchor domain, and wherein the nucleotide sequence that encodes the type II signal anchor domain segment is heterologous with respect to the gene, or gene fragment,
- (b) incubating the recombinant host cells under conditions and a time sufficient for expression of the first member of a complementary/anti-complementary binding pair on the surface of the recombinant cells,
- (c) exposing the recombinant cells to the second member of the complementary/anti-complementary binding pair, and
- (d) selecting recombinant cells that bind the second member of the complementary/anti-complementary binding pair.
5 more elements
A method of selecting nucleic acid molecules that encode a member of a complementary/anti-complementary binding pair, comprising:
- (a) incubating recombinant eukaryotic host cells that comprise an expression vector comprising:
- (i) a eukaryotic promoter,
- (ii) a nucleotide sequence that encodes an intron,
- (iii) a nucleotide sequence encoding a type II signal anchor domain segment, and
- (iv) a nucleotide sequence that encodes an affinity tag,
- (v) a gene, or gene fragment, that encodes the first member of a complementary/anti-complementary binding pair,
- wherein the expression vector comprises elements (i) to (v) in a 5′ to 3′ order, wherein the gene, or gene fragment, resides in-frame with the nucleotide sequence that encodes the type II signal anchor domain, and wherein the nucleotide sequence that encodes the type II signal anchor domain segment is heterologous with respect to the gene, or gene fragment, and wherein the recombinant host cells are incubated under conditions and a time sufficient for expression of the first member of a complementary/anti-complementary binding pair on the surface of the recombinant cells,
- (b) exposing the recombinant cells to the second member of the complementary/anti-complementary binding pair, and
- (c) selecting recombinant cells that bind the second member of the complementary/anti-complementary binding pair.
6 more elements
A method of selecting cells that express a polypeptide of interest from among a collection of cells, comprising:
- (a) incubating a collection of recombinant eukaryotic host cells transfected with a plurality of expression vectors, wherein said plurality of expression vectors comprises a plurality of genes or gene fragments encoding polypeptides and wherein each of said expression vectors comprises:
- (i) a eukaryotic promoter,
- (ii) a nucleotide sequence encoding a type II signal anchor domain segment, and
- (iii) one of said plurality of genes or gene fragments,
- wherein each of said expression vectors comprises elements (i) to (iii) in a 5′ to 3′ order, wherein the one of said plurality of genes or gene fragments resides in-frame with the nucleotide sequence that encodes the type II signal anchor domain, and wherein the nucleotide sequence that encodes the type II signal anchor domain segment is heterologous with respect to the one of said plurality of genes or gene fragments, whereby said plurality of genes or gene fragments are expressed by said cells and whereby said polypeptides are produced as cell-surface polypeptides on said cells, and
- (b) selecting recombinant host cells that produce the polypeptide of interest.
3 more elements
A method of selecting a gene or gene fragment that encodes a polypeptide of interest, comprising:
- (a) transfecting eukaryotic host cells with a plurality of expression vectors to produce a collection of recombinant host cells, wherein said plurality of expression vectors comprises a plurality of genes or gene fragments encoding polypeptides, and wherein each of the expression vectors comprises:
- (i) a eukaryotic promoter,
- (ii) a nucleotide sequence encoding a type II signal anchor domain segment, and
- (iii) one of said plurality of genes or gene fragments,
- wherein each of the expression vectors comprises elements (i) to (iii) in a 5′ to 3′ order, wherein the one of said plurality of genes or gene fragments resides in-frame with the nucleotide sequence that encodes the type II signal anchor domain, and wherein the nucleotide sequence that encodes the type II signal anchor domain segment is heterologous with respect to the one of said plurality of genes or gene fragments;
- (b) incubating the collection of recombinant host cells, whereby each of said cells displays a polypeptide encoded by the one of said plurality of genes or gene fragments;
- and
- (c) selecting recombinant host cells that display the polypeptide of interest on the cell surface.
5 more elements
A method of selecting nucleic acid molecules that encode a polypeptide of interest, comprising:
- (a) incubating recombinant eukaryotic host cells that comprise an expression vector comprising:
- (i) a eukaryotic promoter,
- (ii) a nucleotide sequence that encodes an intron,
- (iii) a nucleotide sequence encoding a type II signal anchor domain segment,
- (iv) a nucleotide sequence that encodes an affinity tag,
- (v) a nucleotide sequence that encodes the polypeptide of interest, and
- wherein the expression vector comprises elements (i) to (v) in a 5′ to 3′ order, wherein the nucleotide sequence that encodes a polypeptide of interest resides in-frame with the nucleotide sequence that encodes the type II signal anchor domain, and wherein the nucleotide sequence that encodes the type II signal anchor domain segment is heterologous with respect to the nucleotide sequence that encodes the polypeptide of interest, and wherein the recombinant host cells are incubated under conditions and a time sufficient for expression of the nucleotide sequence that encodes the polypeptide of interest, and
- (b) selecting recombinant host cells that comprise the polypeptide of interest on the cell surface.
5 more elements
A method of selecting nucleic acid molecules that encode a member of a complementary/anti-complementary binding pair, comprising:
- (a) incubating recombinant eukaryotic host cells that comprise an expression vector comprising:
- (i) a eukaryotic promoter,
- (ii) a nucleotide sequence encoding a type II signal anchor domain segment, and
- (iii) a gene, or gene fragment, that encodes the first member of a complementary/anti-complementary binding pair,
- wherein the expression vector comprises elements (i) to (iii) in a 5′ to 3′ order,
- wherein the gene, or gene fragment, resides in-frame with the nucleotide sequence that encodes the type II signal anchor domain, and wherein the nucleotide sequence that encodes the type II signal anchor domain segment is heterologous with respect to the gene, or gene fragment, and wherein the recombinant host cells are incubated under conditions and a time sufficient for expression of the first member of a complementary/anti-complementary binding pair on the surface of the recombinant cells,
- (b) exposing the recombinant cells to the second member of the complementary/anti-complementary binding pair, and
- (c) selecting recombinant cells that bind the second member of the complementary/anti-complementary binding pair.
5 more elements
Dependencies are inferred from the claim text.