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US5747334A Expired / Lapsed US ⏱️ 0.0 years remaining

Random peptide library

Assignee: University of North Carolina at Chapel Hill
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Filing: 1994-01-31
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Granted: 1998-05-05
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Expires: 2014-01-31
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Abstract

Abstract A novel method for producing novel and/or improved heterofunctional binding fusion proteins termed Totally Synthetic Affinity Reagents (TSARs) is disclosed. TSARs are concatenated heterofunctional proteins, polypeptides or peptides comprising at least two functional regions: a binding domain with affinity for a ligand and a second effector peptide portion that is chemically or biologically active. In one embodiment, the heterofunctional proteins, polypeptides or peptides further comprise a linker peptide portion between the binding domain and the second active peptide portion. The linker peptide can be either susceptible or not susceptible to cleavage by enzymatic or chemical means. Novel and/or improved heterofunctional binding reagents as well as methods for using the reagents for a variety of in vitro and in vivo applications are also disclosed.

Inventors

  • 👤 Brian K Kay
  • 👤 Dana M. Fowlkes
  • 👤 Nils B. Adey
  • 👤 Andrew B. Sparks

Bibliographic Details

Application Number —
Jurisdiction US
Data Source google_patents
Last Synchronized 2026-10-23 16:46
CPC Classifications
C; C40; C40B; C40B40/00; C40B40/02; C07; C07K; C07K14/00; C07K14/435; C07K14/46; C07K14/47; C07K16/00; C07K16/18; C07K16/28; C07K16/30; C07K16/3007; C07K16/3069; C07K16/40; C07K16/44; C12; C12N; C12N15/00; C12N15/09; C12N15/10; C12N15/1034; C12N15/1037; C12N15/1048; C12N15/1062; C12N15/1075; C12N15/11; C12N15/62; C12N15/63; C12N15/70; C07K2319/00; C07K2319/20; C07K2319/50; C07K2319/61; C07K2319/70; C07K2319/705; C07K2319/72; C07K2319/73; C07K2319/735

Claims Analysis

Total Claims: 15
Claim 1
Independent Claim 327 words
A library of recombinant vectors in which each vector encodes one of a plurality of heterofunctional fusion proteins comprising: (a) a binding domain encoded by an oligonucleotide comprising unpredictable nucleotides in which the unpredictable nucleotides are arranged in one or more contiguous sequences, wherein the total number of unpredictable nucleotides is greater than or equal to 60 and less than or equal to 600, wherein the unpredictable nucleotides have the potential to encode all 20 naturally occurring amino acids, and wherein the coding strand of the unpredictable nucleotides comprises the formula (NNB) n+m where N is A, C, G or T; B is G, T or C; and n and m are integers, such that 20≦n+m≦200; wherein the binding domain is encoded by a double stranded oligonucleotide assembled by annealing a first nucleotide sequence of the formula 5' X (NNB) n J Z 3', with a second nucleotide sequence of the formula 3'Z'OU(NNV).sub.m Y5' at corresponding positions Z and Z', where X and Y are restriction enzyme recognition sites, such that X≠Y; N is A, C, G or T; B is G, T or C; V is G, A or C; n is an integer, such that 10≦n≦100; m is an integer, such that 10≦m≦100; Z and Z' are each a sequence of 6, 9 or 12 nucleotides, such that Z and Z' are complementary to each other; and J is A, C, G, T or nothing; O is A, C, G, T or nothing; and U is G, A, C or nothing; provided, however, if any one of J, O or U is nothing then J, O and U are all nothing; and converting the annealed oligonucleotides to a double stranded oligonucleotide; and (b) an effector domain encoded by an oligonucleotide sequence encoding a protein or peptide that enhances expression or detection of the binding domain, said library being capable of being screened to identify a heterofunctional fusion protein having specificity for a ligand of choice.
Claim 2
Dependent Claim 327 words
A library of recombinant vectors in which each vector encodes one of a plurality of heterofunctional fusion proteins comprising: (a) a binding domain encoded by an oligonucleotide comprising unpredictable nucleotides in which the unpredictable nucleotides are arranged in one or more contiguous sequences, wherein the total number of unpredictable nucleotides is greater than or equal to 60 and less than or equal to 600, wherein the unpredictable nucleotides have the potential to encode all 20 naturally occurring amino acids, and wherein the coding strand of the unpredictable nucleotides comprises the formula (NNB) n+m where N is A, C, G or T; B is G, T or C; and n and m are integers, such that 20≦n+m≦200; wherein the binding domain is encoded by a double stranded oligonucleotide assembled by annealing a first nucleotide sequence of the formula 5' X (NNB) n J Z 3', with a second nucleotide sequence of the formula 3'Z'OU(NNV).sub.m Y5' at corresponding positions Z and Z', where X and Y are restriction enzyme recognition sites, such that X≠Y; N is A, C, G or T; B is G, T or C; V is G, A or C; n is an integer, such that 10≦n≦100; m is an integer, such that 10≦m≦100; Z and Z' are each a sequence of 6, 9 or 12 nucleotides, such that Z and Z' are complementary to each other; and J is A, C, G, T or nothing; O is A, C, G, T or nothing; and U is G, A, C or nothing; provided, however, if any one of J, O or U is nothing then J, O and U are all nothing; and converting the annealed oligonucleotides to a double stranded oligonucleotide; and (b) an effector domain encoded by an oligonucleotide sequence encoding a protein or peptide that enhances expression or detection of the binding domain, said library being capable of being screened to identify a heterofunctional fusion protein having specificity for a ligand of choice.
Claim 3
Dependent Claim 17 words
The library according to claim 1, in which the recombinant vectors comprise phage, phagemid or plasmid vectors.
Claim 4
Dependent Claim 28 words
The library according to claim 1, in which the plurality of heterofunctional fusion proteins are expressed on the surface of a phage or viral particle or a cell.
Claim 5
Dependent Claim 25 words
The library according to claim 1, in which the plurality of heterofunctional fusion proteins are expressed and accumulate inside host cells containing the recombinant vectors.
Claim 6
Dependent Claim 23 words
The library according to claim 1, in which the heterofunctional fusion proteins further comprise a linker domain between the binding and effector domains.
Claim 7
Dependent Claim 21 words
The library according to claim 5, in which the linker domain is selectively susceptible to cleavage by chemical or enzymatic means.
Claim 8
Dependent Claim 21 words
The library according to claim 1, in which the binding domains of the heterofunctional fusion proteins can form semirigid conformational structures.
Claim 9
Dependent Claim 39 words
The library according to claim 1, in which each heterofunctional fusion protein comprises at least one disulfide bond forming at least one cystine, thereby allowing for the formation of at least one loop conformation in each heterofunctional fusion protein.
Claim 10
Dependent Claim 39 words
The library according to claim 1, in which each heterofunctional fusion protein comprises at least two disulfide bonds forming at least two cystines, thereby allowing for the formation of at least one cloverleaf-like conformation in each heterofunctional fusion protein.
Claim 11
Dependent Claim 66 words
The library according to claim 1, in which each heterofunctional fusion protein comprises a plurality of both cysteine and histidine residues encoded by nucleotides positioned in or on the flanks of one or more contiguous sequences of unpredictable nucleotides, and in which the positions of the cysteine and histidine residues in the protein are similar to the positions of cysteine and histidine residues in zinc-finger-like proteins.
Claim 12
Dependent Claim 15 words
The library according to claim 1, in which the recombinant vector is a phage vector.
Claim 13
Dependent Claim 22 words
The library according to claim 11 in which the plurality of heterofunctional fusion proteins are displayed on the surface of phage particles.
Claim 14
Dependent Claim 95 words
A library of recombinant vectors in which each vector encodes one of a plurality of heterofunctional fusion proteins comprising: (a) a binding domain encoded by an oligonucleotide comprising unpredictable nucleotides in which the oligonucleotide encodes a polypeptide having the amino acid sequence SSCGX 8 GCGX 8 GCGX 8 GCGPPGSR wherein X is any one of the 20 naturally occurring amino acids; and (b) an effector domain that enhances expression or detection of the binding domain, said library being capable of being screened to identify a heterofunctional fusion protein having specificity for a ligand of choice.
Claim 15
Dependent Claim 95 words
A library of recombinant vectors in which each vector encodes one of a plurality of heterofunctional fusion proteins comprising: (a) a binding domain encoded by an oligonucleotide comprising unpredictable nucleotides in which the oligonucleotide encodes a polypeptide having the amino acid sequence SSCGX 8 GCGX 8 GCGX 8 GCGPPGSR wherein X is any one of the 20 naturally occurring amino acids; and (b) an effector domain that enhances expression or detection of the binding domain, said library being capable of being screened to identify a heterofunctional fusion protein having specificity for a ligand of choice.

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3.5 Years (Year 4) 2001-05-04 2001-11-03 2002-05-05 $2000.00 Window Closed / Lapsed
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