US5770434A
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Soluble peptides having constrained, secondary conformation in solution and method of making same
Assignee: Applied Molecular Evolution Inc
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Filing: 1995-05-15
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Granted: 1998-06-23
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Expires: 2015-05-15
Abstract
Abstract A method of synthesizing isolated, soluble peptides having constrained secondary structure in solution is described herein. The peptides are encoded by expressible oligonucleotides having a desirable bias of random codon sequences.
Inventors
- 👤 William D. Huse
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 04:52
CPC Classifications
C; C40; C40B; C40B40/00; C40B40/02; C07; C07K; C07K14/00; C07K14/005; C12; C12N; C12N15/00; C12N15/09; C12N15/10; C12N15/1034; C12N15/1037; C12N15/63; C12N15/66; C07K2319/00; C12N2795/00; C12N2795/00011; C12N2795/14011; C12N2795/14111; C12N2795/14122; C12N2795/14141; C12N2795/14143
Claims Analysis
Total Claims: 44
Claim 1
Independent Claim
134 words
A composition of matter comprising a plurality of E. coli cells containing a diverse population of expressible oligonucleotides, each of said oligonucleotides encoding a peptide having a desirable bias of predetermined covalent constraints, wherein each of said oligonucleotides is operationally linked to (i) expression elements, (ii) a suppressible stop codon and (iii) a nucleic acid encoding a coat protein contained within an E. coli filamentous bacteriophage vector so as to be expressed as a fusion protein with said coat protein of said E. coli filamentous bacteriophage in a suppressor host or as a secreted peptide in a non-suppressor host, said expressible oligonucleotides having a desirable bias of random codon sequences and wherein said plurality of E. coli cells comprise at least one of said expressible oligonucleotides which encodes a peptide with constrained secondary structure.
Claim 2
Dependent Claim
134 words
A composition of matter comprising a plurality of E. coli cells containing a diverse population of expressible oligonucleotides, each of said oligonucleotides encoding a peptide having a desirable bias of predetermined covalent constraints, wherein each of said oligonucleotides is operationally linked to (i) expression elements, (ii) a suppressible stop codon and (iii) a nucleic acid encoding a coat protein contained within an E. coli filamentous bacteriophage vector so as to be expressed as a fusion protein with said coat protein of said E. coli filamentous bacteriophage in a suppressor host or as a secreted peptide in a non-suppressor host, said expressible oligonucleotides having a desirable bias of random codon sequences and wherein said plurality of E. coli cells comprise at least one of said expressible oligonucleotides which encodes a peptide with constrained secondary structure.
Claim 3
Dependent Claim
23 words
The composition of claim 1, wherein said oligonucleotides have more than one codon encoding an amino acid capable of forming a covalent bond.
Claim 4
Dependent Claim
23 words
The composition of claim 1, wherein said expressible oligonucleotides are expressed as gVIII-fusion proteins on the surface of and E. coli filamentous bacteriophage.
Claim 5
Dependent Claim
26 words
The composition of claim 2, wherein said amino acid is an amino acid selected from the group consisting of cysteine, glutamic acid, lysine, leucine and tyrosine.
Claim 6
Dependent Claim
138 words
A composition of matter comprising a plurality of E. coli cells containing a diverse population of expressible oligonucleotides, each of said oligonucleotides encoding a peptide having a desirable bias of predetermined covalent constraints, wherein each of said oligonucleotides is operationally linked to expression elements and to a nucleic acid encoding a coat protein contained within an E. coli filamentous bacteriophage vector so as to be expressed as a fusion protein or as a secreted peptide, said expressible oligonucleotides having a desirable bias of random codon sequences produced from random combinations of first and second oligonucleotide precursor populations, each or either of said first and second oligonucleotide precursor populations having a desirable bias of random codon sequences and wherein said plurality of cells comprise at least one of said expressible oligonucleotides which encodes a peptide with constrained secondary structure.
Claim 7
Dependent Claim
138 words
A composition of matter comprising a plurality of E. coli cells containing a diverse population of expressible oligonucleotides, each of said oligonucleotides encoding a peptide having a desirable bias of predetermined covalent constraints, wherein each of said oligonucleotides is operationally linked to expression elements and to a nucleic acid encoding a coat protein contained within an E. coli filamentous bacteriophage vector so as to be expressed as a fusion protein or as a secreted peptide, said expressible oligonucleotides having a desirable bias of random codon sequences produced from random combinations of first and second oligonucleotide precursor populations, each or either of said first and second oligonucleotide precursor populations having a desirable bias of random codon sequences and wherein said plurality of cells comprise at least one of said expressible oligonucleotides which encodes a peptide with constrained secondary structure.
Claim 8
Dependent Claim
15 words
The composition of claim 5, wherein said first or second oligonucleotide precursor populations are biased.
Claim 9
Dependent Claim
15 words
The composition of claim 5, wherein said first and second oligonucleotide precursor populations are biased.
Claim 10
Dependent Claim
28 words
The composition of claim 5, wherein said first or second oligonucleotide precursor populations have more than one codon encoding an amino acid capable of forming a covalent bond.
Claim 11
Dependent Claim
28 words
The composition of claim 5, wherein said first and second oligonucleotide precursor populations have at least one codon encoding an amino acid capable of forming a covalent bond.
Claim 12
Dependent Claim
23 words
The composition of claim 5, wherein said expressible oligonucleotides are expressed as gVIII-fusion proteins on the surface of an E. coli filamentous bacteriophage.
Claim 13
Dependent Claim
28 words
The composition of claim 8 or 9, wherein said amino acid is an amino acid selected from the group consisting of cysteine, glutamic acid, lysine, leucine and tyrosine.
Claim 14
Dependent Claim
171 words
A kit for the preparation of vectors for the expression of a peptide:coat protein fusion protein or a secreted peptide from a diverse population of random peptides having a desirable bias of predetermined covalent constraints, wherein at least one of said random peptides in said diverse population is a peptide with constrained secondary structure, said peptides being generated from combined first and second oligonucleotide precursor populations which when combined have a desirable bias of random codon sequences, comprising: two E. coli filamentous bacteriophage vectors, a first filamentous bacteriophage vector having a cloning site for said first precursor oligonucleotides and a pair of restriction sites for operationally combining first precursor oligonucleotides with second precursor oligonucleotides; and a second filamentous bacteriophage vector having a cloning site for said second precursor oligonucleotides and a pair of restriction sites complementary to those on said first vector, one or both vectors containing expression elements capable of being operationally linked to said combined first and second precursor oligonucleotides and to a nucleic acid encoding a coat protein.
Claim 15
Dependent Claim
171 words
A kit for the preparation of vectors for the expression of a peptide:coat protein fusion protein or a secreted peptide from a diverse population of random peptides having a desirable bias of predetermined covalent constraints, wherein at least one of said random peptides in said diverse population is a peptide with constrained secondary structure, said peptides being generated from combined first and second oligonucleotide precursor populations which when combined have a desirable bias of random codon sequences, comprising: two E. coli filamentous bacteriophage vectors, a first filamentous bacteriophage vector having a cloning site for said first precursor oligonucleotides and a pair of restriction sites for operationally combining first precursor oligonucleotides with second precursor oligonucleotides; and a second filamentous bacteriophage vector having a cloning site for said second precursor oligonucleotides and a pair of restriction sites complementary to those on said first vector, one or both vectors containing expression elements capable of being operationally linked to said combined first and second precursor oligonucleotides and to a nucleic acid encoding a coat protein.
Claim 16
Dependent Claim
17 words
The kit of claim 12, wherein said filamentous bacteriophage vectors are derived from M13, f1 or fd.
Claim 17
Dependent Claim
19 words
The kit of claim 12, wherein said first or second oligonucleotide precursor populations are biased toward a pre-determined sequence.
Claim 18
Dependent Claim
19 words
The kit of claim 12, wherein said first and second oligonucleotide precursor populations are biased toward a predetermined sequence.
Claim 19
Dependent Claim
28 words
The kit of claim 12, wherein said first or second oligonucleotide precursor populations have more than one codon encoding an amino acid capable of forming a covalent bond.
Claim 20
Dependent Claim
28 words
The kit of claim 12, wherein said first and second oligonucleotide precursor populations have at least one codon encoding an amino acid capable of forming a covalent bond.
Claim 21
Dependent Claim
28 words
The kit of claim 16 or 17, wherein said amino acid is an amino acid selected from the group consisting of cysteine, glutamic acid, lysine, leucine and tyrosine.
Claim 22
Dependent Claim
140 words
A cloning system for expressing as a peptide:coat protein fusion protein or as a secreted peptide a diverse population of oligonucleotides encoding random peptides having a desirable bias of predetermined covalent constraints, wherein at least one of said oligonucleotides within said diverse population encodes a peptide with constrained secondary structure, said oligonucleotides being generated from a desirable bias of random codon sequences, said cloning system comprising an E. coli filamentous bacteriophage vector having a pair of restriction sites so as to allow the operational combination of said oligonucleotides into a contiguous oligonucleotide encoding said peptide having constrained secondary structure, said contiguous oligonucleotide being operationally linked to expression elements and to a nucleic acid encoding a coat protein contained within an E. coli filamentous bacteriophage so as to be expressed as a peptide:coat protein fusion protein or as a secreted peptide.
Claim 23
Dependent Claim
140 words
A cloning system for expressing as a peptide:coat protein fusion protein or as a secreted peptide a diverse population of oligonucleotides encoding random peptides having a desirable bias of predetermined covalent constraints, wherein at least one of said oligonucleotides within said diverse population encodes a peptide with constrained secondary structure, said oligonucleotides being generated from a desirable bias of random codon sequences, said cloning system comprising an E. coli filamentous bacteriophage vector having a pair of restriction sites so as to allow the operational combination of said oligonucleotides into a contiguous oligonucleotide encoding said peptide having constrained secondary structure, said contiguous oligonucleotide being operationally linked to expression elements and to a nucleic acid encoding a coat protein contained within an E. coli filamentous bacteriophage so as to be expressed as a peptide:coat protein fusion protein or as a secreted peptide.
Claim 24
Dependent Claim
24 words
The cloning system of claim 19, wherein said oligonucleotides have more than one codon encoding an amino acid capable of forming a covalent bond.
Claim 25
Dependent Claim
170 words
A cloning system for expressing as a peptide:coat protein fusion protein or as a secreted peptide a diverse population of oligonucleotides encoding random peptides having a desirable bias of predetermined covalent constraints wherein at least one of said oligonucleotides within said diverse population encodes a peptide with constrained secondary structure, said oligonucleotides being generated from diverse populations of combined first and second oligonucleotide precursor populations each or either having a desirable bias of random codon sequences said cloning system, comprising: a set of first filamentous bacteriophage vectors having a diverse population of first precursor oligonucleotides having a desirable bias of random codon sequences and a second set of filamentous bacteriophage vectors having a diverse population of second precursor oligonucleotides having a desirable bias of random codon sequences, said first and second vectors each having a pair of restriction sites so as to allow the operational combination of said oligonucleotides into a contiguous oligonucleotide encoding said peptide having constrained secondary structure and to a nucleic acid encoding a bacteriophage coat protein.
Claim 26
Dependent Claim
170 words
A cloning system for expressing as a peptide:coat protein fusion protein or as a secreted peptide a diverse population of oligonucleotides encoding random peptides having a desirable bias of predetermined covalent constraints wherein at least one of said oligonucleotides within said diverse population encodes a peptide with constrained secondary structure, said oligonucleotides being generated from diverse populations of combined first and second oligonucleotide precursor populations each or either having a desirable bias of random codon sequences said cloning system, comprising: a set of first filamentous bacteriophage vectors having a diverse population of first precursor oligonucleotides having a desirable bias of random codon sequences and a second set of filamentous bacteriophage vectors having a diverse population of second precursor oligonucleotides having a desirable bias of random codon sequences, said first and second vectors each having a pair of restriction sites so as to allow the operational combination of said oligonucleotides into a contiguous oligonucleotide encoding said peptide having constrained secondary structure and to a nucleic acid encoding a bacteriophage coat protein.
Claim 27
Dependent Claim
15 words
The composition of claim 21, wherein said first or second oligonucleotide precursor populations are biased.
Claim 28
Dependent Claim
15 words
The composition of claim 21, wherein said first and second oligonucleotide precursor populations are biased.
Claim 29
Dependent Claim
29 words
The cloning system of claim 21, wherein said first or second oligonucleotide precursor populations have more than one codon encoding an amino acid capable of forming a covalent bond.
Claim 30
Dependent Claim
29 words
The cloning system of claim 21, wherein said first and second oligonucleotide precursor populations have at least one codon encoding an amino acid capable of forming a covalent bond.
Claim 31
Dependent Claim
20 words
The cloning system of claim 21, wherein said first and second vectors are combined through a pair of restriction sites.
Claim 32
Dependent Claim
23 words
The composition of claim 21, wherein said expressible oligonucleotides are expressed as gVIII-fusion proteins on the surface of an E. coli filamentous bacteriophage.
Claim 33
Dependent Claim
11 words
An expressible oligonucleotide produced by the cloning system of claim 21.
Claim 34
Dependent Claim
10 words
A host cell containing the cloning system of claim 21.
Claim 35
Dependent Claim
29 words
The cloning system of claim 24 or 25, wherein said amino acid is an amino acid selected from the group consisting of cysteine, glutamic acid, lysine, leucine and tyrosine.
Claim 36
Dependent Claim
87 words
A population of vectors comprising a diverse population of oligonucleotides operationally linked to (i) expression elements, (ii) a suppressible stop codon and (iii) a nucleic acid encoding a coat protein so as to be expressed as a fusion protein with said coat protein of an E. coli filamentous bacteriophage in a suppressor host or as a secreted peptide in a non-suppressor host, said oligonucleotides having a desirable bias of random codon sequences, and more than one codon encoding an amino acid capable of forming a covalent bond.
Claim 37
Dependent Claim
87 words
A population of vectors comprising a diverse population of oligonucleotides operationally linked to (i) expression elements, (ii) a suppressible stop codon and (iii) a nucleic acid encoding a coat protein so as to be expressed as a fusion protein with said coat protein of an E. coli filamentous bacteriophage in a suppressor host or as a secreted peptide in a non-suppressor host, said oligonucleotides having a desirable bias of random codon sequences, and more than one codon encoding an amino acid capable of forming a covalent bond.
Claim 38
Dependent Claim
28 words
A population of vectors of claim 31, wherein said amino acid is an amino acid selected from the group consisting of cysteine, glutamic acid, lysine, leucine and tyrosine.
Claim 39
Dependent Claim
11 words
A host cell containing the population of vectors of claim 32.
Claim 40
Dependent Claim
40 words
A method of isolating a soluble peptide having a desirable bias of predetermined covalent constraints, comprising growing said host cell of claim 29 or 33 under suitable conditions favoring expression of said peptide, and affinity isolating said peptide so produced.
Claim 41
Independent Claim
190 words
A method of constructing a diverse population of vectors containing combined first and second oligonucleotide precursor populations, wherein each or either precursor oligonucleotides has a desirable bias of random codon sequences, and which express said combined oligonucleotides which encode random peptides having a desirable bias of predetermined covalent constraints, wherein at least one of said peptides exhibits constrained secondary structure, comprising the steps of: (a) operationally linking sequences from a diverse population of first precursor oligonucleotides having a desirable bias of random codon sequences to a first E. coli filamentous bacteriophage vector; (b) operationally linking sequences from a diverse population of second precursor oligonucleotides having a desirable bias of random codon sequences to a second E. coli filamentous bacteriophage vectors, wherein said first or second, or first and second precursor oligonucleotides have at least one codon capable of forming a covalent bond; and (c) combining the vector products of steps (a) and (b) under conditions where said populations of first and second precursor oligonucleotides are joined together into a population of combined vectors which express said random peptides as a fusion with a coat protein or as a secreted peptide.
Claim 42
Independent Claim
190 words
A method of constructing a diverse population of vectors containing combined first and second oligonucleotide precursor populations, wherein each or either precursor oligonucleotides has a desirable bias of random codon sequences, and which express said combined oligonucleotides which encode random peptides having a desirable bias of predetermined covalent constraints, wherein at least one of said peptides exhibits constrained secondary structure, comprising the steps of: (a) operationally linking sequences from a diverse population of first precursor oligonucleotides having a desirable bias of random codon sequences to a first E. coli filamentous bacteriophage vector; (b) operationally linking sequences from a diverse population of second precursor oligonucleotides having a desirable bias of random codon sequences to a second E. coli filamentous bacteriophage vectors, wherein said first or second, or first and second precursor oligonucleotides have at least one codon capable of forming a covalent bond; and (c) combining the vector products of steps (a) and (b) under conditions where said populations of first and second precursor oligonucleotides are joined together into a population of combined vectors which express said random peptides as a fusion with a coat protein or as a secreted peptide.
Claim 43
Dependent Claim
26 words
The method of claim 35, wherein said amino acid is an amino acid selected from the group consisting of cysteine, glutamic acid, lysine, leucine and tyrosine.
Claim 44
Dependent Claim
16 words
The method of claim 35, wherein steps (a) through (d) are repeated two or more times.
References Cited (Backward Citations)
US4458066A
Cited
US4771000A
Cited
US5185147A
Cited
US5223409A
Cited
US5403484A
Cited
EP0383620A2
Cited
US5427908A
Cited
US5270163A
Cited
US5432018A
Cited
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