High throughput generation and screening of fully human antibody repertoire in yeast
First Claim
1. A kit, comprising:
- a first and second populations of haploid yeast cells of opposite mating types, the first population of haploid yeast cells comprising a library of tester expression vectors for the library of tester fusion proteins, each of the tester expression vector comprising a first transcription sequence encoding either an activation domain or a DNA binding domain of a transcription activator, a first nucleotide sequence encoding a first polypeptide subunit, a second nucleotide sequence encoding a second polypeptide subunit, and a linker sequence encoding a linker peptide that links the first nucleotide sequence and the second nucleotide sequence, wherein the first polypeptide subunit, the linker peptide, the second polypeptide are expressed as a tester fusion protein with either the activation domain or the DNA binding domain of the transcription activator;
the second population of haploid yeast cells comprises a target expression vector for a target fusion protein, the target expression vector comprising a second transcription sequence encoding either the activation domain or the DNA binding domain of the transcription activator which is not expressed by the library of tester expression vectors, and a target sequence encoding the target protein or peptide, wherein the target protein or peptide is expressed as a fusion protein with either the activation domain or the DNA binding domain of the transcription activator which is not expressed by the library of tester expression vectors, and either the first or second population of haploid yeast cells comprises a reporter construct comprising a reporter gene whose expression is under transcriptional control of the transcription activator.
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Abstract
Compostions, kits and methods are provided for generating highly diverse libraries of proteins such as antibodies via homologous recombination in vivo, and screening these libraries against protein, peptide and nucleic acid targets using a two-hybrid method in yeast. The method for screening a library of tester proteins against a target protein or peptide comprises: expressing a library of tester proteins in yeast cells, each tester protein being a fusion protein comprised of a first polypeptide subunit whose sequence varies within the library, a second polypeptide subunit whose sequence varies within the library independently of the first polypeptide, and a linker peptide which links the first and second polypeptide subunits; expressing one or more target fusion proteins in the yeast cells expressing the tester proteins, each of the target fusion proteins comprising a target peptide or protein; and selecting those yeast cells in which a reporter gene is expressed, the expression of the reporter gene being activated by binding of the tester fusion protein to the target fusion protein.
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Citations
55 Claims
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1. A kit, comprising:
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a first and second populations of haploid yeast cells of opposite mating types, the first population of haploid yeast cells comprising a library of tester expression vectors for the library of tester fusion proteins, each of the tester expression vector comprising a first transcription sequence encoding either an activation domain or a DNA binding domain of a transcription activator, a first nucleotide sequence encoding a first polypeptide subunit, a second nucleotide sequence encoding a second polypeptide subunit, and a linker sequence encoding a linker peptide that links the first nucleotide sequence and the second nucleotide sequence, wherein the first polypeptide subunit, the linker peptide, the second polypeptide are expressed as a tester fusion protein with either the activation domain or the DNA binding domain of the transcription activator;
the second population of haploid yeast cells comprises a target expression vector for a target fusion protein, the target expression vector comprising a second transcription sequence encoding either the activation domain or the DNA binding domain of the transcription activator which is not expressed by the library of tester expression vectors, and a target sequence encoding the target protein or peptide, wherein the target protein or peptide is expressed as a fusion protein with either the activation domain or the DNA binding domain of the transcription activator which is not expressed by the library of tester expression vectors, and either the first or second population of haploid yeast cells comprises a reporter construct comprising a reporter gene whose expression is under transcriptional control of the transcription activator. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
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15. A kit, comprising:
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a first and second populations of haploid yeast cells of opposite mating types, the first population of haploid yeast cells comprising a library of tester expression vectors for the library of tester fusion proteins, each of the tester expression vector comprising a first transcription sequence encoding either an activation domain or a DNA binding domain of a transcription activator, a first nucleotide sequence encoding a first polypeptide subunit, a second nucleotide sequence encoding a second polypeptide subunit, and a linker sequence encoding a linker peptide that links the first nucleotide sequence and the second nucleotide sequence, wherein the first polypeptide subunit, the linker peptide, the second polypeptide are expressed as a tester fusion protein with either the activation domain or the DNA binding domain of the transcription activator, wherein either the first or second population of haploid yeast cells comprises a reporter construct comprising a reporter gene whose expression is under transcriptional control of the transcription activator. - View Dependent Claims (16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26)
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27. A method for generating a library of yeast expression vectors, comprising:
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a) transforming into yeast cells i) a linearized yeast expression vector having a 5′
- and 3′
-terminus sequence at a first site of linearization; and
ii) a library of first insert nucleotide sequences that are linear, double stranded, each of the first insert sequences comprising a first nucleotide sequence encoding a first polypeptide subunit, a 5′
- and 3′
-flanking sequence at the ends of the first insert sequence which are sufficiently homologous to the 5′
- and 3′
-terminus sequences of the vector at the first site of linearization, respectively, to enable homologous recombination to occur;
b) having homologous recombination occur between the linearized yeast expression vector and the first insert sequence in the transformed yeast cells, such that the first insert sequence is included in the expression vector;
c) isolating from the transformed yeast cells the expression vectors that contain the library of the first insert sequences;
d) linearizing the expression vectors containing the library of the first insert sequences to generate a 5′
- and 3′
-terminus sequence at a second site of linearization;
e) transforming into yeast cells i) the linearized expression vectors in step d), and ii) a library of second insert nucleotide sequences that are linear, double stranded, each of the second insert sequences comprising a second nucleotide sequence encoding a second polypeptide subunit, a 5′
- and 3′
-flanking sequence at the ends of the second insert sequence which are sufficiently homologous to the 5′
- and 3′
-terminus sequences of the vector at the second site of linearization, respectively, to enable homologous recombination to occur; and
f) having homologous recombination occur between the linearized yeast expression vector at the second linearization site and the second insert sequence in the transformed yeast cells, such that the second insert sequence is included in the vector and the first and second nucleotide sequences are linked by a linker sequence;
wherein the expression vector expresses the first polypeptide subunit, the second polypeptide subunit, and the linker polypeptide as a single fusion protein in the transformed yeast cells by the library of yeast expression vectors; and
the first and second nucleotide sequences each independently varies within the library of expression vectors. - View Dependent Claims (28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55)
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Specification