US5723286A
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Peptide library and screening systems
Assignee: Affymax Technologies NV
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Filing: 1990-06-20
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Granted: 1998-03-03
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Expires: 2010-06-20
Abstract
Abstract Peptides which bind to selected receptor molecules are identified by screening libraries which encode a random or controlled collection of amino acids. Peptides encoded by the libraries are expressed as fusion proteins of bacteriophage coat proteins, and bacteriophage particles are then screened against the receptors of interest. Peptides having a wide variety of uses, such as therapeutic or diagnostic reagents, may thus be identified without any prior information on the structure of the expected ligand or receptor molecule.
Inventors
- 👤 William J. Dower
- 👤 Steven E. Cwirla
- 👤 Ronald W. Barrett
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-05 21:32
CPC Classifications
C; C40; C40B; C40B30/00; C40B30/04; C07; C07K; C07K1/00; C07K1/04; C07K1/047; C07K14/00; C07K14/435; C07K14/665; C07K14/675; C07K5/00; C07K5/04; C07K5/08; C07K5/0802; C07K5/0804; C07K5/0806; C07K5/0812; C07K5/10; C07K5/1002; C07K5/1005; C07K5/1008; C07K5/1016; C12; C12N; C12N15/00; C12N15/09; C12N15/10; C12N15/1034; C12N15/1037; C12N15/11; C12N15/62; C12N15/63; C12N15/70; C12Q; C12Q1/00; C12Q1/70; C40B40/00; C40B40/02; G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/68; G01N33/6803; G01N33/6845; G01N33/6878; C07K2319/00
Claims Analysis
Total Claims: 49
Claim 1
Independent Claim
184 words
A method for identifying a peptide which binds to a preselected receptor molecule, comprising: transforming host cells by electroporation with at least 10 8 different bacteriophage expression vectors, wherein each of said different vectors encodes a fusion protein comprising a peptide fused to a coat protein of a filamentous bacteriophage so that the N-terminal amino acid of said fusion protein is the N-terminal amino acid of said peptide, and wherein said different vectors are constructed by ligating each oligonucleotide of a mixture of at least 10 8 different oligonucleotides to a bacteriophage cloning vector that encodes the coat protein so as to form a coding sequence for said fusion protein, wherein each of said different oligonucleotides comprises a series of codons encoding a random collection of amino acids and encodes a different peptide; cultivating the transformed cell under conditions suitable for expression and assembly of bacteriophage particles thereby displaying said peptides on the surface of said particles; and selecting bacteriophage particles displaying the peptide by combining said particles with the preselected receptor molecule and separating particles bound to said receptor molecule from unbound particles.
Claim 2
Independent Claim
184 words
A method for identifying a peptide which binds to a preselected receptor molecule, comprising: transforming host cells by electroporation with at least 10 8 different bacteriophage expression vectors, wherein each of said different vectors encodes a fusion protein comprising a peptide fused to a coat protein of a filamentous bacteriophage so that the N-terminal amino acid of said fusion protein is the N-terminal amino acid of said peptide, and wherein said different vectors are constructed by ligating each oligonucleotide of a mixture of at least 10 8 different oligonucleotides to a bacteriophage cloning vector that encodes the coat protein so as to form a coding sequence for said fusion protein, wherein each of said different oligonucleotides comprises a series of codons encoding a random collection of amino acids and encodes a different peptide; cultivating the transformed cell under conditions suitable for expression and assembly of bacteriophage particles thereby displaying said peptides on the surface of said particles; and selecting bacteriophage particles displaying the peptide by combining said particles with the preselected receptor molecule and separating particles bound to said receptor molecule from unbound particles.
Claim 3
Dependent Claim
23 words
The method of claim 1, further comprising the step of sequencing a region of said expression vectors comprising said oligonucleotides of selected bacteriophage.
Claim 4
Dependent Claim
34 words
The method of claim 1, wherein a pool of said selected bacteriophage particles is enriched by repeating the selection step at least once, wherein the selected bacteriophage particles are propagated between said selection steps.
Claim 5
Dependent Claim
24 words
The method of claim 1, where the receptor is bound to a solid phase and the selected bacteriophage particles are separated from the culture.
Claim 6
Dependent Claim
16 words
The method of claim 4, wherein said receptor molecule is an antibody or binding fragment thereof.
Claim 7
Dependent Claim
14 words
The method of claim 1, wherein the filamentous bacteriophage is f1, fd, or M13.
Claim 8
Dependent Claim
14 words
The method of claim 6, wherein the bacteriophage is fd or a derivative thereof.
Claim 9
Dependent Claim
15 words
The method of claim 7, wherein the coat protein of the fd bacteriophage is pIII.
Claim 10
Dependent Claim
47 words
The method of claim 1, wherein the codons encoding the collection of amino acids are represented by (NNK) x or (NNS) x , where N is A, C, G, or T, K is G or T, S is G or C, and x is at least 7.
Claim 11
Dependent Claim
9 words
The method of claim 9, wherein x is eight.
Claim 12
Dependent Claim
16 words
The method of claim 10, wherein at least 10% of the recombinant bacteriophage particles are screened.
Claim 13
Dependent Claim
19 words
The method of claim 11, wherein each of said 10 8 different oligonucleotides encodes at least one spacer residue.
Claim 14
Dependent Claim
11 words
The method of claim 12, wherein said spacer residue is Gly.
Claim 15
Dependent Claim
13 words
The method of claim 12, wherein said oligonucleotides encode a spacer comprising Gly-Gly.
Claim 16
Dependent Claim
28 words
The method of claim 12, wherein a codon encoding a spacer residue is at one terminus of said series of codons encoding said random collection of amino acids.
Claim 17
Dependent Claim
23 words
The method of claim 15, wherein said codon encodes a Cys residue and is present on both terminii of said series of codons.
Claim 18
Dependent Claim
40 words
The method of claim 12, wherein said oligonucleotides have the formula (NNK) y (abc) n (NNK) z ; wherein a, b, and c are A, G, C, or T, (y+z) is at least 7, and n is at least 1.
Claim 19
Dependent Claim
10 words
The method of claim 17, wherein (abc) n encodes Gly-Pro-Gly.
Claim 20
Dependent Claim
24 words
The method of claim 1, wherein said series of codons of said 10 8 different oligonucleotides is prepared from a condensation of activated trinucleotides.
Claim 21
Dependent Claim
39 words
The method of claim 1, wherein said fusion protein is a preprotein which is processed by the host cell so that the N-terminal amino acid of said peptide is the N-terminal amino acid of said fusion protein after processing.
Claim 22
Dependent Claim
33 words
The method according to claim 20, wherein the fusion protein further comprises spacer amino acid residues between the N-terminal amino acid of the coat protein and the C-terminal amino acid of said peptide.
Claim 23
Dependent Claim
26 words
The method of claim 21 wherein said fusion protein further comprises a second peptide between the spacer and the N-terminal amino acid of the coat protein.
Claim 24
Dependent Claim
11 words
The method of claim 20, wherein said coat protein is pIII.
Claim 25
Dependent Claim
28 words
The method of claim 1, wherein each of said 10 8 different bacteriophage expression vectors is a double stranded circle of nucleic acid with a single stranded gap.
Claim 26
Dependent Claim
19 words
The method of claim 1, wherein each of said 10 8 different oligonucleotides is a double stranded nucleic acid.
Claim 27
Dependent Claim
20 words
The method of claim 1, wherein each of said 10 8 different oligonucleotides is a partially single stranded nucleic acid.
Claim 28
Dependent Claim
30 words
The method of claim 26, wherein said partially single stranded oligonucleotides are rendered completely double stranded subsequent to insertion into said bacteriophage cloning vector and prior to said transforming step.
Claim 29
Dependent Claim
36 words
The method of claim 26, wherein each of said 10 8 different oligonucleotides is composed of first, second, and third single stranded oligonucleotides that are annealed to one another prior to insertion into said cloning vector.
Claim 30
Dependent Claim
64 words
The method of claim 28, wherein said first single stranded oligonucleotide comprises a first series of codons encoding said peptide flanked on one side by a second series of codons encoding at least a portion of a signal peptide for said bacteriophage coat protein, and on another side by a third series of codons encoding at least a portion of said bacteriophage coat protein.
Claim 31
Dependent Claim
33 words
The method of claim 29, wherein said first series of codons is (NNK) x ; N is A, G, C, or T; K is G or T; and x is at least 7.
Claim 32
Dependent Claim
14 words
The method of claim 30, wherein said first series of codons is single stranded.
Claim 33
Dependent Claim
27 words
The method of claim 1, wherein said oligonucleotides are inserted into said bacteriophage cloning vector to replace a segment bounded by two unique, non-complementary restriction enzyme sites.
Claim 34
Dependent Claim
18 words
The method of claim 32, wherein said non-complementary restriction enzyme sites are recognized by a single restriction enzyme.
Claim 35
Dependent Claim
11 words
The method of claim 33, wherein said restriction enzyme is BstXI.
Claim 36
Dependent Claim
20 words
The method of claim 1, wherein said cloning vector does not produce active, infectious phage particles in a host cell.
Claim 37
Dependent Claim
47 words
The method of claim 1, wherein said selected bacteriophage particles are sequenced to ascertain a specific sequence encoded by selected particles, and a second set of bacteriophage expression vectors is constructed, wherein each member of said second set of vectors encodes a variation of said specific sequence.
Claim 38
Dependent Claim
29 words
The method of claim 36, further comprising the steps of isolating said expression vectors from said selected bacteriophage in single stranded form; and mutagenizing said single stranded expression vectors.
Claim 39
Dependent Claim
27 words
The method of claim 37, wherein said mutagenizing step comprises treatment with a chemical agent selected from the group consisting of nitric acid, formic acid, and hydrazine.
Claim 40
Independent Claim
227 words
A method for identifying a peptide which binds to a preselected receptor molecule, comprising: transforming host cells by electroporation with at least 10 8 different bacteriophage expression vectors, wherein each of said different vectors encodes a fusion protein comprising a peptide fused to a coat protein of a filamentous bacteriophage, and wherein said different vectors differ from each other with respect to the peptide of said fusion protein encoded by said vector; cultivating the transformed cell under conditions suitable for expression and assembly of bacteriophage particles thereby displaying said peptides on the surface of said particles; and selecting bacteriophage particles displaying the peptide by combining said particles with the preselected receptor molecule and separating particles bound to said receptor molecule from unbound particles wherein each of said 10 8 different oligonucleotides is composed of first, second, and third single-stranded oligonucleotides that are annealed to one another prior to insertion into said cloning vector; and wherein said first single stranded oligonucleotide is 5' C T C T C A C T C C (NNK) x A C T G T T G A A A G T T G T; and said second and third single stranded oligonucleotides are 5' G G A G T G A G A G T A G A and 5' C T T T C A A C A G T, respectively.
Claim 41
Independent Claim
227 words
A method for identifying a peptide which binds to a preselected receptor molecule, comprising: transforming host cells by electroporation with at least 10 8 different bacteriophage expression vectors, wherein each of said different vectors encodes a fusion protein comprising a peptide fused to a coat protein of a filamentous bacteriophage, and wherein said different vectors differ from each other with respect to the peptide of said fusion protein encoded by said vector; cultivating the transformed cell under conditions suitable for expression and assembly of bacteriophage particles thereby displaying said peptides on the surface of said particles; and selecting bacteriophage particles displaying the peptide by combining said particles with the preselected receptor molecule and separating particles bound to said receptor molecule from unbound particles wherein each of said 10 8 different oligonucleotides is composed of first, second, and third single-stranded oligonucleotides that are annealed to one another prior to insertion into said cloning vector; and wherein said first single stranded oligonucleotide is 5' C T C T C A C T C C (NNK) x A C T G T T G A A A G T T G T; and said second and third single stranded oligonucleotides are 5' G G A G T G A G A G T A G A and 5' C T T T C A A C A G T, respectively.
Claim 42
Independent Claim
157 words
A method for identifying a peptide which binds to a preselected receptor molecule, comprising: transforming host cells by electroporation with at least 10 8 different bacteriophage expression vectors, wherein each of said different vectors encodes a fusion protein comprising a peptide fused to a coat protein of a filamentous bacteriophage, and wherein said different vectors differ from each other with respect to the peptide of said fusion protein encoded by said vector; cultivating the transformed cell under conditions suitable for expression and assembly of bacteriophage particles thereby displaying said peptides on the surface of said particles; and selecting bacteriophage particles displaying the peptide by combining said particles with the preselected receptor molecule and separating particles bound to immobilized preselected receptor molecule from unbound particles; repeating the selection step at least once, wherein the selected bacteriophage particles are propagated between said selection steps wherein said receptor is immobilized at reduced densities in subsequent repetitions of the selecting step.
Claim 43
Independent Claim
157 words
A method for identifying a peptide which binds to a preselected receptor molecule, comprising: transforming host cells by electroporation with at least 10 8 different bacteriophage expression vectors, wherein each of said different vectors encodes a fusion protein comprising a peptide fused to a coat protein of a filamentous bacteriophage, and wherein said different vectors differ from each other with respect to the peptide of said fusion protein encoded by said vector; cultivating the transformed cell under conditions suitable for expression and assembly of bacteriophage particles thereby displaying said peptides on the surface of said particles; and selecting bacteriophage particles displaying the peptide by combining said particles with the preselected receptor molecule and separating particles bound to immobilized preselected receptor molecule from unbound particles; repeating the selection step at least once, wherein the selected bacteriophage particles are propagated between said selection steps wherein said receptor is immobilized at reduced densities in subsequent repetitions of the selecting step.
Claim 44
Dependent Claim
24 words
The method of claim 40, wherein in one of the selecting steps said preselected receptor is present at a density favoring monovalent binding interactions.
Claim 45
Dependent Claim
30 words
The method of claim 41, wherein bacteriophage particles displaying high affinity peptides are selected based on a slow dissociation rate between said preselected receptor molecules and said high affinity peptides.
Claim 46
Dependent Claim
12 words
The method of claim 40, wherein said preselected receptor molecule is monovalent.
Claim 47
Independent Claim
141 words
A method for identifying a peptide which binds to a preselected receptor molecule, comprising: transforming host cells by electroporation with at least 10 8 different bacteriophage expression vectors, wherein each of said different vectors encodes a fusion protein comprising a peptide fused to a coat protein of a filamentous bacteriophage, and wherein said different vectors differ from each other with respect to the peptide of said fusion protein encoded by said vector; cultivating the transformed cell under conditions suitable for expression and assembly of bacteriophage particles thereby displaying said peptides on the surface of said particles; and selecting bacteriophage particles displaying the peptide by combining said particles with the preselected receptor molecule and separating particles bound to said receptor molecule from unbound particles, wherein said bacteriophage displaying the peptide are combined with said preselected receptor molecule under conditions favoring monovalent binding.
Claim 48
Independent Claim
141 words
A method for identifying a peptide which binds to a preselected receptor molecule, comprising: transforming host cells by electroporation with at least 10 8 different bacteriophage expression vectors, wherein each of said different vectors encodes a fusion protein comprising a peptide fused to a coat protein of a filamentous bacteriophage, and wherein said different vectors differ from each other with respect to the peptide of said fusion protein encoded by said vector; cultivating the transformed cell under conditions suitable for expression and assembly of bacteriophage particles thereby displaying said peptides on the surface of said particles; and selecting bacteriophage particles displaying the peptide by combining said particles with the preselected receptor molecule and separating particles bound to said receptor molecule from unbound particles, wherein said bacteriophage displaying the peptide are combined with said preselected receptor molecule under conditions favoring monovalent binding.
Claim 49
Dependent Claim
21 words
The method of claim 44, wherein said peptide is within about 100 amino acids of an N-terminus of said fusion protein.
References Cited (Backward Citations)
US4593002A
Cited
GB2183661A
Cited
US4833092A
Cited
WO1988005085A1
Cited
WO1988006630A1
Cited
WO1989006694A1
Cited
US4910140A
Cited
US5223409A
Cited
WO1990002809A1
Cited
WO1990005144A1
Cited
WO1990014424A1
Cited
WO1992001047A1
Cited
WO1992007077A1
Cited
WO1992009690A2
Cited
WO1992015679A1
Cited
WO1992015677A1
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
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| 3.5 Years (Year 4) | 2001-03-02 | 2001-09-01 | 2002-03-03 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2005-03-02 | 2005-09-01 | 2006-03-03 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2009-03-02 | 2009-09-01 | 2010-03-03 | $7700.00 | Window Closed / Lapsed |