US5723323A
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Method of identifying a stochastically-generated peptide, polypeptide, or protein having ligand binding property and compositions thereof
Assignee: Individual
•
Filing: 1994-12-02
•
Granted: 1998-03-03
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Expires: 2014-12-02
Abstract
Abstract The present invention is directed to a process for the production of a peptide, polypeptide, or protein having a predetermined property. In accordance with one embodiment, the process begins by producing by way of synthetic polynucleotide coupling, stochastically generated polynucleotide sequences. A library of expression vectors containing such stochastically generated polynucleotide sequences is formed. Next, host cells containing the vectors are cultured so as to produce peptides, polypeptides, or proteins encoded by the stochastically generated polynucleotide sequences. Screening or selection is carried out on such host cells to identify a peptide, polypeptide, or protein produced by the host cells which has the predetermined property. The stochastically generated polynucleotide sequence which encodes the identified peptide, polypeptide, or protein is then isolated and used to produce the peptide, polypeptide, or protein having the predetermined property.
Inventors
- 👤 Stuart Alan Kauffman
- 👤 Marc Ballivet
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 23:52
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/10; C12N15/1034; C12N15/1093; C07; C07K; C07K14/00; C07K14/005; C07K14/435; C07K14/475; C07K14/485; C12N15/1048; C12N15/63; C12N15/64; C12N15/66; C12N15/67; C12N15/68; C12N15/70; C12N9/00; C12N9/14; C12N9/24; C12N9/2402; C12N9/2468; C12N9/2471; C12Y; C12Y302/00; C12Y302/01; C12Y302/01023; A; A61; A61K; A61K39/00; C07K2319/00; C12N2730/00; C12N2730/00011; C12N2730/10011; C12N2730/10111; C12N2730/10122
Claims Analysis
Total Claims: 59
Claim 1
Independent Claim
124 words
A method of identifying a peptide, polypeptide or protein having a binding property to a ligand, comprising: (a) providing a ligand for detecting said binding property; (b) synthesizing a diverse population of stochastically generated polynucleotide sequences; (c) inserting said diverse population of stochastically generated polynucleotide sequences into a population of expression vectors to form a diverse population of expression vectors containing stochastically generated polynucleotide sequences; (d) expressing in host cells said diverse population of expression vectors containing stochastically generated polynucleotide sequences to produce a diverse population of peptides, polypeptides or proteins; and (e) screening said diverse population of peptides, polypeptides or proteins with said ligand under conditions which allow binding and detection of one or more peptides, polypeptides or proteins having said predetermined property.
Claim 2
Independent Claim
124 words
A method of identifying a peptide, polypeptide or protein having a binding property to a ligand, comprising: (a) providing a ligand for detecting said binding property; (b) synthesizing a diverse population of stochastically generated polynucleotide sequences; (c) inserting said diverse population of stochastically generated polynucleotide sequences into a population of expression vectors to form a diverse population of expression vectors containing stochastically generated polynucleotide sequences; (d) expressing in host cells said diverse population of expression vectors containing stochastically generated polynucleotide sequences to produce a diverse population of peptides, polypeptides or proteins; and (e) screening said diverse population of peptides, polypeptides or proteins with said ligand under conditions which allow binding and detection of one or more peptides, polypeptides or proteins having said predetermined property.
Claim 3
Dependent Claim
23 words
The method of claim 1, wherein said stochastically generated polynucleotide sequences further comprises all twenty amino acid residues encoded at each codon position.
Claim 4
Dependent Claim
23 words
The method of claim 1, wherein said diverse population of stochastically generated polynucleotide sequences, encode at least 10,000 different peptides, polypeptides or proteins.
Claim 5
Dependent Claim
14 words
The method of claim 1, wherein said inserting further comprises hybridization of complementary ends.
Claim 6
Dependent Claim
11 words
The method of claim 1, wherein said inserting further comprises ligation.
Claim 7
Dependent Claim
23 words
The method of claim 1, wherein said diverse population of stochastically generated polynucleotide sequences are produced by stochastic copolymerization of double stranded oligonucleotides.
Claim 8
Dependent Claim
32 words
The method of claim 1, wherein said diverse population of stochastically generated polynucleotide sequences are produced by copolymerization of the four kinds of nucleotide triphosphates consisting of Adenine, Cytosine, Guanine and Thymine.
Claim 9
Dependent Claim
19 words
The method of claim 1, wherein said diverse population of stochastically generated polynucleotide sequences are produced by chemical synthesis.
Claim 10
Dependent Claim
12 words
The method of claim 1, wherein the expression vector is a plasmid.
Claim 11
Dependent Claim
10 words
The method of claim 9, wherein the plasmid is pUC8.
Claim 12
Dependent Claim
12 words
The method of claim 1, wherein the expression vector is vital DNA.
Claim 13
Dependent Claim
17 words
The method of claim 1, wherein the expression vector is a hybrid of plasmid and vital DNA.
Claim 14
Dependent Claim
12 words
The method of claim 1, wherein the expression vector is a phage.
Claim 15
Dependent Claim
55 words
The method of claim 1, wherein step (c) further comprises digesting the diverse population of expression vectors with a restriction enzyme having a recognition sequence absent in the expression vector and reinserting the digested products into said digested population of vectors to form a different population having a greater number of new stochastic polynucleotide sequences.
Claim 16
Dependent Claim
25 words
An isolated, diverse population of peptides, polypeptides, or proteins comprising greater than about 1×10 5 different stochastic amino acid sequences encoded by stochastic polynucleotide sequences.
Claim 17
Dependent Claim
25 words
An isolated, diverse population of peptides, polypeptides, or proteins comprising greater than about 1×10 5 different stochastic amino acid sequences encoded by stochastic polynucleotide sequences.
Claim 18
Independent Claim
152 words
A method of isolating a polynucleotide sequence encoding a peptide, polypeptide or protein having a predetermined binding property to a ligand, comprising: (a) providing a ligand for detecting said binding property; (b) synthesizing a diverse population of stochastically generated polynucleotide sequences; (c) inserting said diverse population of stochastically generated polynucleotide sequences into a population of expression vectors to form a diverse population of expression vectors containing stochastically generated polynucleotide sequences; (d) expressing in host cells said diverse population of expression vectors containing stochastically generated polynucleotide sequences to produce a diverse population of peptides, polypeptides or proteins; (e) screening said diverse population of peptides, polypeptides or proteins with said ligand under conditions which allow binding and detection of one or more peptides, polypeptides or proteins to said ligand; and (f) isolating the stochastically generated polynucleotide sequence or sequences which encoding said peptides, polypeptides or proteins having said predetermined binding property to said ligand.
Claim 19
Independent Claim
152 words
A method of isolating a polynucleotide sequence encoding a peptide, polypeptide or protein having a predetermined binding property to a ligand, comprising: (a) providing a ligand for detecting said binding property; (b) synthesizing a diverse population of stochastically generated polynucleotide sequences; (c) inserting said diverse population of stochastically generated polynucleotide sequences into a population of expression vectors to form a diverse population of expression vectors containing stochastically generated polynucleotide sequences; (d) expressing in host cells said diverse population of expression vectors containing stochastically generated polynucleotide sequences to produce a diverse population of peptides, polypeptides or proteins; (e) screening said diverse population of peptides, polypeptides or proteins with said ligand under conditions which allow binding and detection of one or more peptides, polypeptides or proteins to said ligand; and (f) isolating the stochastically generated polynucleotide sequence or sequences which encoding said peptides, polypeptides or proteins having said predetermined binding property to said ligand.
Claim 20
Dependent Claim
23 words
The method of claim 16, wherein said stochastically generated polynucleotide sequences further comprises all twenty amino acid residues encoded at each codon position.
Claim 21
Dependent Claim
23 words
The method of claim 16, wherein said diverse population of stochastically generated polynucleotide sequences, encode at least 10,000 different peptides, polypeptides or proteins.
Claim 22
Dependent Claim
14 words
The method of claim 16, wherein said inserting further comprises hybridization of complementary ends.
Claim 23
Dependent Claim
11 words
The method of claim 16, wherein said inserting further comprises ligation.
Claim 24
Dependent Claim
23 words
The method of claim 16, wherein said diverse population of stochastically generated polynucleotide sequences are produced by stochastic copolymerization of double stranded oligonucleotides.
Claim 25
Dependent Claim
32 words
The method of claim 16, wherein said diverse population of stochastically generated polynucleotide sequences are produced by copolymerization of the four kinds of nucleotide triphosphates consisting of Adenine, Cytosine, Guanine and Thymine.
Claim 26
Dependent Claim
55 words
The method of claim 16, wherein step (c) further comprises digesting the diverse population of expression vectors with a restriction enzyme having a recognition sequence absent in the expression vector and reinserting the digested products into said digested population of vectors to form a different population having a greater number of new stochastic polynucleotide sequences.
Claim 27
Dependent Claim
36 words
An isolated, diverse population of polynucleotide sequences which encode a diverse population of ligand binding peptides, polypeptides, or proteins comprising greater than about 1×10 5 different stochastic amino acid sequences encoded by the stochastic polynucleotide sequences.
Claim 28
Dependent Claim
36 words
An isolated, diverse population of polynucleotide sequences which encode a diverse population of ligand binding peptides, polypeptides, or proteins comprising greater than about 1×10 5 different stochastic amino acid sequences encoded by the stochastic polynucleotide sequences.
Claim 29
Independent Claim
165 words
A method of isolating a peptide, polypeptide or protein having a binding property, comprising: (a), providing a ligand for detecting said binding property; (b) synthesizing a diverse population of stochastically generated polynucleotide sequences; (c) inserting said diverse population of stochastically generated polynucleotide sequences into a population of expression vectors to form a diverse population of expression vectors containing stochastically generated polynucleotide sequences; (d) expressing in host cells said diverse population of expression vectors containing stochastically generated polynucleotide sequences to produce a diverse population of peptides, polypeptides or proteins; (e) screening said diverse population of peptides, polypeptides or proteins with said ligand under conditions which allow binding and detection of one or more peptides, polypeptides or proteins having said predetermined property; (f) isolating the stochastically generated polynucleotide sequence or sequences encoding said peptides, polypeptides or proteins having said binding property to said ligand; and (g) using genetic information from said isolated stochastically generated polynucleotide sequence to produce said peptide, polypeptide or protein having said binding property.
Claim 30
Independent Claim
165 words
A method of isolating a peptide, polypeptide or protein having a binding property, comprising: (a), providing a ligand for detecting said binding property; (b) synthesizing a diverse population of stochastically generated polynucleotide sequences; (c) inserting said diverse population of stochastically generated polynucleotide sequences into a population of expression vectors to form a diverse population of expression vectors containing stochastically generated polynucleotide sequences; (d) expressing in host cells said diverse population of expression vectors containing stochastically generated polynucleotide sequences to produce a diverse population of peptides, polypeptides or proteins; (e) screening said diverse population of peptides, polypeptides or proteins with said ligand under conditions which allow binding and detection of one or more peptides, polypeptides or proteins having said predetermined property; (f) isolating the stochastically generated polynucleotide sequence or sequences encoding said peptides, polypeptides or proteins having said binding property to said ligand; and (g) using genetic information from said isolated stochastically generated polynucleotide sequence to produce said peptide, polypeptide or protein having said binding property.
Claim 31
Dependent Claim
23 words
The method of claim 25, wherein said stochastically generated polynucleotide sequences further comprises all twenty amino acid residues encoded at each codon position.
Claim 32
Dependent Claim
23 words
The method of claim 25, which said diverse population of stochastically generated polynucleotide sequences, encode at least 10,000 different peptides, polypeptides or proteins.
Claim 33
Dependent Claim
14 words
The method of claim 25, wherein said inserting further comprises hybridization of complementary ends.
Claim 34
Dependent Claim
11 words
The method of claim 25, wherein said inserting further comprises ligation.
Claim 35
Dependent Claim
23 words
The method of claim 25, wherein said diverse population of stochastically generated polynucleotide sequences are produced by stochastic copolymerization of double stranded oligonucleotides.
Claim 36
Dependent Claim
32 words
The method of claim 25, wherein said diverse population of stochastically generated polynucleotide sequences are produced by copolymerization of the four kinds of nucleotide triphosphates consisting of Adenine, Cytosine, Guanine and Thymine.
Claim 37
Dependent Claim
19 words
The method of claim 25, wherein said diverse population of stochastically generated polynucleotide sequences are produced by chemical synthesis.
Claim 38
Dependent Claim
56 words
The method of claim 25, wherein step (c) further comprises digesting the diverse population of expression vectors with a restriction enzyme having a recognition sequence absent in the expression vector and reinserting the digested products into said digested population of vectors to form a new different population having a greater number of new stochastic polynucleotide sequences.
Claim 39
Independent Claim
92 words
A method of producing a diverse population of stochastically generated polynucleotide sequences encoding a diverse population of ligand binding peptides, polypeptides or proteins, comprising: (a) synthesizing a diverse population of stochastically generated polynucleotide sequences selected from a method consisting of stochastic copolymerization of double stranded oligonucleotides, copolymerization of the four kinds of nucleotide triphoshpates consisting of Adenine, Cytosine, Guanine and Thymine, and chemical synthesis; and (b) inserting said diverse population of stochastically generated polynucleotide sequences into a population of vectors to form a diverse population of vectors containing stochastically generated polynucleotide sequences.
Claim 40
Independent Claim
92 words
A method of producing a diverse population of stochastically generated polynucleotide sequences encoding a diverse population of ligand binding peptides, polypeptides or proteins, comprising: (a) synthesizing a diverse population of stochastically generated polynucleotide sequences selected from a method consisting of stochastic copolymerization of double stranded oligonucleotides, copolymerization of the four kinds of nucleotide triphoshpates consisting of Adenine, Cytosine, Guanine and Thymine, and chemical synthesis; and (b) inserting said diverse population of stochastically generated polynucleotide sequences into a population of vectors to form a diverse population of vectors containing stochastically generated polynucleotide sequences.
Claim 41
Dependent Claim
23 words
The method of claim 34, wherein said stochastically generated polynucleotide sequences further comprises all twenty amino acid residues encoded at each codon position.
Claim 42
Dependent Claim
23 words
The method of claim 34, which said diverse population of stochastically generated polynucleotide sequences, encode at least 10,000 different peptides, polypeptides or proteins.
Claim 43
Dependent Claim
14 words
The method of claim 34, wherein said inserting further comprises hybridization of complementary ends.
Claim 44
Dependent Claim
11 words
The method of claim 34, wherein said inserting further comprises ligation.
Claim 45
Dependent Claim
21 words
The method of claim 34, further comprising introducing said diverse population of vectors containing stochastically generated polynucleotide sequences into host cells.
Claim 46
Dependent Claim
56 words
The method of claim 34, wherein step (b) further comprises digesting the diverse population of expression vectors with a restriction enzyme having a recognition sequence absent in the expression vector and reinserting the digested products into said digested population of vectors to form a new different population having a greater number of new stochastic polynucleotide sequences.
Claim 47
Dependent Claim
33 words
An isolated, diverse population of vectors comprising stochastically generated polynucleotide sequences encoding a diverse population of ligand binding peptides, polypeptides, or proteins comprising greater than about 1×10 5 different stochastic amino acid sequences.
Claim 48
Dependent Claim
33 words
An isolated, diverse population of vectors comprising stochastically generated polynucleotide sequences encoding a diverse population of ligand binding peptides, polypeptides, or proteins comprising greater than about 1×10 5 different stochastic amino acid sequences.
Claim 49
Dependent Claim
26 words
An isolated, diverse population of peptides or polypeptides comprising stochastic amino acid sequences encoded by stochastic polynucleotide sequences of about 300 nucleotides or less in length.
Claim 50
Dependent Claim
26 words
An isolated, diverse population of peptides or polypeptides comprising stochastic amino acid sequences encoded by stochastic polynucleotide sequences of about 300 nucleotides or less in length.
Claim 51
Dependent Claim
35 words
An isolated, diverse population of polynucleotides sequences encoding a diverse population of ligand binding peptides or polypeptides comprising stochastic amino acid sequences encoded by stochastic polynucleotide sequences of about 300 nucleotides or less in length.
Claim 52
Dependent Claim
35 words
An isolated, diverse population of polynucleotides sequences encoding a diverse population of ligand binding peptides or polypeptides comprising stochastic amino acid sequences encoded by stochastic polynucleotide sequences of about 300 nucleotides or less in length.
Claim 53
Dependent Claim
35 words
An isolated, diverse population of vectors encoding a diverse population of ligand binding peptides or polypeptides comprising stochastic amino acid sequences encoded by stochastically generated polynucleotide sequences of about 300 nucleotides or less in length.
Claim 54
Dependent Claim
35 words
An isolated, diverse population of vectors encoding a diverse population of ligand binding peptides or polypeptides comprising stochastic amino acid sequences encoded by stochastically generated polynucleotide sequences of about 300 nucleotides or less in length.
Claim 55
Dependent Claim
65 words
An isolated, diverse population of peptides, polypeptides, or proteins comprising stochastic amino acid sequences produced by a method comprising synthesizing a diverse population of stochastically generated polynucleotide sequences selected from a method consisting of stochastic copolymerization of double stranded oligonucleotides, copolymerization of the four kinds of nucleotide triphosphates consisting of Adenine, Cytosine, Guanine and Thymine, and chemical synthesis; and expressing said stochastically generated polynucleotide sequences.
Claim 56
Dependent Claim
65 words
An isolated, diverse population of peptides, polypeptides, or proteins comprising stochastic amino acid sequences produced by a method comprising synthesizing a diverse population of stochastically generated polynucleotide sequences selected from a method consisting of stochastic copolymerization of double stranded oligonucleotides, copolymerization of the four kinds of nucleotide triphosphates consisting of Adenine, Cytosine, Guanine and Thymine, and chemical synthesis; and expressing said stochastically generated polynucleotide sequences.
Claim 57
Dependent Claim
24 words
The isolated diverse population of peptides, polypeptides, or proteins of claim 15, further comprising greater than about 1×10 6 different stochastic amino acid sequences.
Claim 58
Dependent Claim
24 words
The isolated diverse population of peptides, polypeptides, or proteins of claim 15, further comprising greater than about 1×10 7 different stochastic amino acid sequences.
Claim 59
Dependent Claim
24 words
The isolated, diverse population of peptides, polypeptides, or proteins of claim 15, further comprising greater than about 1×10 8 different stochastic amino acid sequences.
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