US5338665A
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Peptide library and screening method
Assignee: Affymax Technologies NV
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Filing: 1992-10-15
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Granted: 1994-08-16
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Expires: 2012-10-15
Abstract
Abstract A random peptide library constructed by transforming host cells with a collection of recombinant vectors that encode a fusion protein comprised of a DNA binding protein and a random peptide and also contain a binding site for the DNA binding protein can be used to screen for novel ligands. The screening method results in the formation of a complex comprising the fusion protein bound to a receptor through the random peptide ligand and to the recombinant DNA vector through the DNA binding protein.
Inventors
- 👤 Peter J. Schatz
- 👤 Willem P. C. Stemmer
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 09:30
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/68; C; C12; C12N; C12N15/00; C12N15/09; C12N15/10; C12N15/1034; C12N15/1075
Claims Analysis
Total Claims: 17
Claim 1
Independent Claim
147 words
A method for generating and screening a random peptide library, said method comprising the steps of: (a) transforming host cells with at least 10 6 different vectors, each of which (i) encodes a fusion protein composed of a random peptide linked to a DNA binding protein consisting essentially of two lac headpieces joined by a linker; and (ii) contains a lacO or a derivative lacO binding site, and culturing the resultant transformed host cells under conditions such that said fusion protein is produced and binds to a vector that encodes the fusion protein; (b) lysing the cells transformed in step (a) under conditions such that the fusion protein remains bound to the vector that encodes the fusion protein; (c) contacting the fusion proteins with a receptor under conditions conducive to specific peptide-receptor binding; and (d) isolating the vector that encodes a peptide that binds to said receptor.
Claim 2
Independent Claim
147 words
A method for generating and screening a random peptide library, said method comprising the steps of: (a) transforming host cells with at least 10 6 different vectors, each of which (i) encodes a fusion protein composed of a random peptide linked to a DNA binding protein consisting essentially of two lac headpieces joined by a linker; and (ii) contains a lacO or a derivative lacO binding site, and culturing the resultant transformed host cells under conditions such that said fusion protein is produced and binds to a vector that encodes the fusion protein; (b) lysing the cells transformed in step (a) under conditions such that the fusion protein remains bound to the vector that encodes the fusion protein; (c) contacting the fusion proteins with a receptor under conditions conducive to specific peptide-receptor binding; and (d) isolating the vector that encodes a peptide that binds to said receptor.
Claim 3
Dependent Claim
19 words
The method of claim 1, wherein said random peptide is located at the carboxy terminus of said fusion protein.
Claim 4
Dependent Claim
36 words
The method of claim 1 further comprising the steps of: (e) transforming host cells with the vectors obtained in step (d); and repeating steps (b), (c), and (d) with the host cells transformed in step (e).
Claim 5
Dependent Claim
12 words
The method of claim 3, wherein said host cells are E. coli.
Claim 6
Dependent Claim
13 words
The method of claim 4, wherein said binding site is lacO S .
Claim 7
Dependent Claim
17 words
The method of claim 4, wherein said vector is a plasmid that contains only one binding site.
Claim 8
Dependent Claim
114 words
A mixture of recombinant host cells transformed with a random peptide library composed of at least 10 6 different members, wherein each member is a recombinant DNA vector that (i) encodes a DNA binding protein consisting essentially of two lac headpieces joined by a linker; (ii) contains a lacO or derivative lacO binding site; and (iii) comprises a coding sequence for a random peptide inserted into the coding sequence of the DNA binding protein such that the resulting vector encodes a fusion protein that is composed of the DNA binding protein and the random peptide; and wherein each different member differs from other members with respect to the coding sequence of the random peptide.
Claim 9
Dependent Claim
114 words
A mixture of recombinant host cells transformed with a random peptide library composed of at least 10 6 different members, wherein each member is a recombinant DNA vector that (i) encodes a DNA binding protein consisting essentially of two lac headpieces joined by a linker; (ii) contains a lacO or derivative lacO binding site; and (iii) comprises a coding sequence for a random peptide inserted into the coding sequence of the DNA binding protein such that the resulting vector encodes a fusion protein that is composed of the DNA binding protein and the random peptide; and wherein each different member differs from other members with respect to the coding sequence of the random peptide.
Claim 10
Dependent Claim
113 words
A mixture of recombinant host cells transformed with a ligand fragment library composed of at least 10 different members, wherein each member is a recombinant DNA vector that (i) encodes a DNA binding protein consisting essentially of two lac headpieces joined by a linker; (ii) contains a lacO or derivative lacO binding site; and (iii) comprises a coding sequence for a ligand fragment inserted into the coding sequence of the DNA binding protein, such that the resulting vector encodes a fusion protein that is composed of the DNA binding protein and the ligand fragment; and wherein each different member differs from other members with respect to the coding sequence of the ligand fragment.
Claim 11
Dependent Claim
113 words
A mixture of recombinant host cells transformed with a ligand fragment library composed of at least 10 different members, wherein each member is a recombinant DNA vector that (i) encodes a DNA binding protein consisting essentially of two lac headpieces joined by a linker; (ii) contains a lacO or derivative lacO binding site; and (iii) comprises a coding sequence for a ligand fragment inserted into the coding sequence of the DNA binding protein, such that the resulting vector encodes a fusion protein that is composed of the DNA binding protein and the ligand fragment; and wherein each different member differs from other members with respect to the coding sequence of the ligand fragment.
Claim 12
Dependent Claim
93 words
A recombinant DNA vector comprising: (a) a DNA sequence encoding a DNA binding protein consisting essentially of two lac headpieces joined by a linker; (b) a promoter positioned so as to drive transcription of said DNA binding protein coding sequence; (c) a lacO or derivative lacO binding site; and (d) a coding sequence for a peptide inserted in said DNA binding protein coding sequence so that said coding sequences can be transcribed to produce a fusion protein composed of said peptide and said DNA binding protein capable of binding to said binding site.
Claim 13
Dependent Claim
93 words
A recombinant DNA vector comprising: (a) a DNA sequence encoding a DNA binding protein consisting essentially of two lac headpieces joined by a linker; (b) a promoter positioned so as to drive transcription of said DNA binding protein coding sequence; (c) a lacO or derivative lacO binding site; and (d) a coding sequence for a peptide inserted in said DNA binding protein coding sequence so that said coding sequences can be transcribed to produce a fusion protein composed of said peptide and said DNA binding protein capable of binding to said binding site.
Claim 14
Dependent Claim
13 words
The vector of claim 9, wherein said binding site is lacO S .
Claim 15
Dependent Claim
8 words
The vector of claim 9 that is pDimer1.
Claim 16
Dependent Claim
13 words
A recombinant E. coli host cell transformed with a vector of claim 9.
Claim 17
Dependent Claim
11 words
The transformed host cell of claim 12 that is E. coli/pDimer1.
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1997-08-15 | 1998-02-14 | 1998-08-16 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2001-08-15 | 2002-02-14 | 2002-08-16 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2005-08-15 | 2006-02-14 | 2006-08-16 | $7700.00 | Window Closed / Lapsed |