US 5,498,538 AGranted

Totally synthetic affinity reagents

AssigneeThe University of North Carolina at Chapel Hill

InventorsBrian K. Kay, Dana M. Fowlkes

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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.

Claims26

Dependent claims

A method for identifying a protein, polypeptide and/or peptide which binds to a ligand of choice, comprising:

screening a library of recombinant vectors in which each vector encodes one of a plurality of heterofunctional fusion proteins containing
(a) a binding domain encoded by a double stranded oligonucleotide assembled by annealing a first nucleotide sequence of the formula 5' X (NNB).sub.n J Z 3' with a second nucleotide sequence of the formula
3'Z'O U (NNV).sub.m Y5'
where
X and Y are restriction enzyme recognition sites, such that X.noteq.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.ltoreq.n.ltoreq.100;
m is an integer, such that 10.ltoreq.m.ltoreq.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, by contacting the plurality of heterofunctional fusion proteins with said ligand of choice under conditions conducive to ligand binding and isolating any fusion prosthesis which bind said ligand.

16 more elements

A method for identifying a protein and/or peptide which binds to a ligand of choice, comprising:

(a) generating a library of vectors in which each vector encodes one of a plurality of heterofunctional fusion proteins containing
(i) a binding domain encoded by a double stranded oligonucleotide assembled by annealing a first nucleotide sequence of the formula 5' X (NNB).sub.n J Z 3' with a second nucleotide sequence of the formula
3'Z'O U (NNV)m Y 5'
where
X and Y are restriction enzyme recognition sites, such that X.noteq.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.ltoreq.n.ltoreq.100;
m is an integer, such that 10.ltoreq.m.ltoreq.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,
(ii) an effector domain encoded by an oligonucleotide sequence encoding a protein or peptide that enhances expression or detection of the binding domain;
and
(b) screening the library of vectors by contacting the plurality of heterofunctional fusion proteins with said ligand of choice under conditions conducive to ligand binding and isolating any heterofunctional fusion protein which binds said ligand.

20 more elements

Dependencies are inferred from the claim text.

Facts

Application: 08/176,500Filed 30 Dec 1993 · continuation
Priority: 15 Feb 1990
Granted: 12 Mar 1996
Term ends: Not estimatedThe source records don’t support an estimate.

Classification26 codes

C07K 14/47
From vertebrates: from mammals
C07K 16/00
Immunoglobulins [ig]
C07K 16/18
Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies: against material from animals or humans
C07K 16/3007
From tumour cells: carcino-embryonic Antigens