US 2003/0207290 A1 · Methods, reagents, kits and apparatus for protein function analysis

Abstract

Apparatus, systems, system components, methods, compositions, and reagents for determining the function and activity of peptides and proteins and for identifying and characterizing molecules that affect these functions and activities. More specifically, methods and reagents used to determine the various activities of peptides and proteins including their binding specificity, binding activity, their enzymatic activity and their ability to act as substrates for enzymes. The disclosed methods are especially suited for the analysis of peptide and protein function where large numbers of peptides and proteins need to be analyzed.

Claims132

Dependent claims

A multi-well plate with 20 or more wells containing the in vitro transcription and translation products of unique nucleic acid constructs.

A multi-well plate with 20 or more wells coated with a binding species and containing the in vitro transcription and translation products of unique nucleic acid constructs, said products being immobilized in said wells via binding interactions with said binding species.

A multi-well plate with 20 or more wells containing the in vitro transcription and translation products of unique nucleic acid constructs, said products being immobilized in said wells via non-specific adsorption.

A multi-well plate with at least 20 wells containing in vitro transcription and translation products of unique nucleic acid constructs, each of said wells produced by the method comprising:

a) obtaining a nucleic acid construct,
b) transcribing said nucleic acid to generate RNA,
c) translating said RNA in a cell-free translation system containing a tRNA precharged with a modified amino acid, to produce an amino acid sequence containing said modified amino acid, and
d) contacting said amino acid sequence with a surface of a solid phase present in said well so as to immobilize said amino acid sequence on said surface.

1 more element

A multi-well plate for screening for an activity of interest, said plate having 20 or more wells containing the in vitro transcription and translation products of unique nucleic acid constructs and said wells having immobilized therein assay domains comprising an assay reagent selected from the group consisting of i) a substrate of said activity, ii) a binding reagent specific for a substrate of said activity and iii) a binding reagent specific for a product of said activity.

A method of producing a multi-well plate with at least 20 wells containing unique in vitro transcription and translation products, said method comprising, for each of said wells:

a) obtaining a nucleic acid construct,
b) transcribing said nucleic acid construct to generate RNA,
c) translating said RNA in a cell-free translation system containing a tRNA precharged with a modified amino acid, to produce an amino acid sequence containing said modified amino acid, and
d) contacting said amino acid sequence with a surface of a solid phase present in said well so as to immobilize said amino acid sequence on said surface.

1 more element

A method for screening nucleic acid constructs for those encoding amino acid sequences subject to a post-translational modification activity comprising the following steps:

a) obtaining a nucleic acid construct,
b) transcribing said nucleic acid to generate RNA,
c) translating said RNA in a cell-free system containing a tRNA precharged with a modified amino acid, to produce an amino acid sequence containing said modified amino acid, and
d) detecting said post-translational modification of said amino acid sequence by a solid phase binding assay for the binding of said amino acid sequence to a binding species specific for said post-translational modification.

1 more element

A method for screening nucleic acid constructs for those encoding amino acid sequences subject to a post-translational modification activity comprising the following steps:

(a) obtaining a nucleic acid construct,
(b) transcribing said nucleic acid to generate RNA,
(c) translating said RNA in a cell-free system containing a tRNA precharged with a modified amino acid and a substrate that can be linked to a protein to form a post-translational modification, said substrate being modified with a detectable species, to produce an amino acid sequence containing said modified amino acid and said post-translational modification, and
(d) detecting said post-translational modification of said amino acid sequence by a solid phase binding assay for the binding of said amino acid sequence to a binding species specific for said post-translational modification or the binding of said amino acid sequence to a binding reagent specific for said modified amino acid.

1 more element

A method for screening nucleic acid constructs for those encoding amino acid sequences with enzymatic activity comprising the following steps:

a) obtaining a nucleic acid construct,
b) transcribing said nucleic acid to generate RNA,
c) translating said RNA in a cell-free system containing a tRNA precharged with a modified amino acid, to produce an amino acid sequence containing said modified amino acid, and
d) detecting enzymatic activity of said amino acid sequence using a method comprising the following steps:
i) capturing said amino acid sequence onto a solid phase via an immobilized binding reagent specific for said modified amino acid,
ii) adding enzyme reaction buffer, and
iii) detecting the product of the enzyme activity.

4 more elements

A method for screening nucleic acid constructs for those encoding amino acid sequences that are substrates of enzymatic activity comprising the following steps:

a) obtaining a nucleic acid constructs,
b) transcribing said nucleic acid to generate RNA,
c) translating said RNA in a cell-free system containing a tRNA precharged with a modified amino acid, to produce an amino acid sequence containing said modified amino acid, and
d) detecting said amino acid sequences which are substrates of an enzyme activity using a method comprising the following steps:
i) capturing said amino acid sequence onto a solid phase via an immobilized binding reagent specific for said modified amino acid;
ii) adding an enzyme;
and
iii) detecting the product of the enzyme activity on said amino acid sequence.

5 more elements

A method for screening nucleic acid constructs for those encoding amino acid sequences with binding activity comprising the following steps:

a) obtaining a nucleic acid construct,
b) transcribing said nucleic acid to generate RNA,
c) translating said RNA in a cell-free system containing a tRNA precharged with a modified amino acid, to produce an amino acid sequence containing said modified amino acid, and
d) detecting binding activity of said amino acid sequence for a binding partner via a solid phase binding assay.

1 more element

A method for screening nucleic acid constructs for those encoding amino acid sequences with binding activity comprising the following steps:

a) obtaining a first nucleic acid construct and second nucleic acid construct,
b) transcribing said first nucleic acid construct to generate a first RNA and transcribing said second nucleic acid constructs to generate a second RNA,
c) translating said first RNA in a cell-free system containing a tRNA precharged with an amino acid containing a binding species, to produce a first amino acid sequence containing said binding species,
d) translating said second RNA in a cell-free system containing a tRNA precharged with an amino acid containing a detectable species, to produce a second amino acid sequence containing said detectable species, and
e) detecting binding activity of said first amino acid sequence to said second amino acid sequence using a method comprising the following steps:
i) contacting a sample of said first amino acid sequence with said second amino acid sequence, and
ii) capturing said binding species on a surface of a well of a multi-well plate via a second binding species
iii) detecting said detectable species bound to said surface.

5 more elements

A method for screening nucleic acid constructs for those encoding amino acid sequences with nascent binding activity comprising the following steps:

a) obtaining a nucleic acid construct,
b) transcribing said nucleic acid to generate RNA,
c) translating said RNA in a cell-free system containing a tRNA precharged with a modified amino acid, to produce an amino acid sequence containing said modified amino acid,
d) capturing said amino acid sequence on a surface of a well of a multi-well plate via a binding reagent specific for said modified amino acid to form a coated surface,
e) contacting said amino acid sequence with a modifying activity to covalently modify said amino acid sequence,
f) contacting said modified amino acid sequence with a second binding species labeled with a detectable species, and
g) detecting the complex of said second binding species and said immobilized amino acid sequence.

4 more elements

A method for screening for antigens with binding activity for antibodies comprising the following steps:

(a) obtaining a nucleic acid construct,
(b) transcribing said nucleic acid in vitro to generate RNA,
(c) translating said RNA in a cell-free system, to produce an amino acid sequence, and
(d) conducting a solid phase binding assay to detect the binding of said amino acid sequence with an antibody.

1 more element

The method of any one of claims 33-79 or 104, wherein said solid phase is a bead in a well of a multi-well plate.

The method of any one of claims 33-79, 104 or 105, wherein said solid phase is a surface of a well of a multi-well plate.

A method for screening nucleic acid constructs for those encoding amino acid sequences subject to a post-translational modification activity comprising the following steps:

a) obtaining a nucleic acid construct coding for an amino acid sequence and an affinity tag,
b) transcribing said nucleic acid to generate RNA,
c) translating said RNA in a cell-free system to produce an amino acid sequence linked to said affinity tag, and
d) detecting said post-translational modification of said amino acid sequence by a solid phase binding assay for the binding of said amino acid sequence to a binding species specific for said post-translational modification.

1 more element

A method for screening nucleic acid constructs for those encoding amino acid sequences subject to a post-translational modification activity comprising the following steps:

(a) obtaining a nucleic acid construct coding for an amino acid sequence and an affinity tag,
(b) transcribing said nucleic acid to generate RNA,
(c) translating said RNA in a cell-free system containing a substrate that can be linked to a protein to form a post-translational modification, said substrate being modified with a detectable species, to produce an amino acid sequence containing said affinity tag and said post-translational modification, and
(d) detecting said post-translational modification of said amino acid sequence by a solid phase binding assay for the binding of said amino acid sequence to a binding species specific for said post-translational modification or the binding of said amino acid sequence to a binding reagent specific for said affinity tag.

1 more element

A method for screening nucleic acid constructs for those encoding amino acid sequences with enzymatic activity comprising the following steps:

(a) obtaining a nucleic acid construct coding for an amino acid sequence and an affinity tag,
(b) transcribing said nucleic acid to generate RNA,
(c) translating said RNA in a cell-free system to produce an amino acid sequence linked to said affinity tag, and
(d) detecting enzymatic activity of said amino acid sequence using a method comprising the following steps:
i) capturing said amino acid sequence onto a solid phase via an immobilized binding reagent specific for said affinity tag,
ii) adding enzyme reaction buffer, and
ii) detecting the product of the enzyme activity.

4 more elements

A method for screening nucleic acid constructs for those encoding amino acid sequences that are substrates of enzymatic activity comprising the following steps:

(a) obtaining a nucleic acid construct coding for an amino acid sequence and an affinity tag,
(b) transcribing said nucleic acid to generate RNA,
(c) translating said RNA in a cell-free system to produce an amino acid sequence containing said modified amino acid, and
(d) detecting said amino acid sequences which are substrates of an enzyme activity using a method comprising the following steps:
i) capturing said amino acid sequence onto a solid phase via an immobilized binding reagent specific for said affinity tag,
ii) adding an enzyme;
and
iii) detecting the product of the enzyme activity on said amino acid sequence.

5 more elements

A method for screening nucleic acid constructs for those encoding amino acid sequences with binding activity comprising the following steps:

(a) obtaining a nucleic acid construct coding for an amino acid sequence and an affinity tag,
(b) transcribing said nucleic acid to generate RNA,
(c) translating said RNA in a cell-free system to produce an amino acid sequence containing said modified amino acid, and
(d) detecting binding activity of said amino acid sequence for a binding partner via a solid phase binding assay.

1 more element

A method for screening nucleic acid constructs for those encoding amino acid sequences with binding activity comprising the following steps:

(a) obtaining a first nucleic acid construct and a second nucleic acid construct, said nucleic acid constructs coding for first and second amino acid sequences linked to first and second affinity tags,
(b) transcribing said first nucleic acid construct to generate a first RNA and transcribing said second nucleic acid constructs to generate a second RNA,
(c) translating said first RNA in a cell-free system to produce a first amino acid sequence linked to said first affinity tag,
(d) translating said second RNA in a cell-free system to produce a second amino acid sequence linked to said second affinity tag, and
(e) detecting binding activity of said first amino acid sequence to said second amino acid sequence using a method comprising the following steps:
i) contacting a sample of said first amino acid sequence with said second amino acid sequence,
ii) capturing said first affinity tag on a surface of a well of a multi-well plate via a first binding species, and
iii) detecting said second affinity tag bound to said surface.

5 more elements

A method for screening nucleic acid constructs for those encoding amino acid sequences with nascent binding activity comprising the following steps:

(a) obtaining a nucleic acid construct coding for an amino acid sequence linked to an affinity tag,
(b) transcribing said nucleic acid to generate RNA,
(c) translating said RNA in a cell-free system to produce an amino acid sequence linked to said affinity tag,
(d) capturing said amino acid sequence on a surface of a well of a multi-well plate via a binding reagent specific for said affinity tag to form a coated surface,
(e) contacting said amino acid sequence with a modifying activity to covalently modify said amino acid sequence,
(f) contacting said modified amino acid sequence with a second binding species labeled with a detectable species, and
(g) detecting the complex of said second binding species and said immobilized amino acid sequence.

4 more elements

A method for screening nucleic acid constructs for those encoding amino acid sequences with enzymatic activity comprising the following steps:

(a) obtaining a nucleic acid construct,
(b) transcribing said nucleic acid to generate RNA,
(c) translating said RNA in a cell-free system to produce an amino acid sequence, and
(d) detecting enzymatic activity of said amino acid sequence using a method comprising the following steps:
i) contacting said amino acid sequence with an enzyme substrate immobilized on a solid phase, and
ii) measuring conversion of the substrate to product.

3 more elements

A method for measuring the kinase activity of amino acid sequences coded by nucleic acid constructs comprising the following steps:

(a) obtaining a nucleic acid construct,
(b) transcribing said nucleic acid to generate RNA,
(c) translating said RNA in a cell-free system containing a tRNA precharged with a modified amino acid, to produce an amino acid sequence containing said modified amino acid, and
(d) detecting enzymatic activity of said amino acid sequence using a method comprising the following steps:
i) capturing said amino acid sequence on a solid phase via an immobilized binding reagent specific for said modified amino acid,
ii) incubating said protein under conditions suitable for kinase activity and optionally, comprising a soluble kinase substrate, and
iii) measuring the tyrosine kinase activity of said amino acid sequence.

4 more elements

An in vitro kinase assay for a kinase selected from the list comprising:

kit oncogene, fibroblast growth factor receptor 2, endothelial-specific receptor tyrosine kinase, B-cell src-homology tyrosine kinase, Eph receptor B3, Janus kinase 1 (a protein tyrosine kinase), fibroblast growth factor receptor 1 (fms-related TK2), FYN oncogene related to SRC, hemopoietic cell kinase, CDC like kinase 4, Yamaguchi sarcoma viral (v-yes) oncogene homolog, CDC-like kinase 2, Eph receptor B4, CDC-like kinase 3, fibroblast growth factor receptor 1, kinase insert domain protein receptor, fibroblast growth factor receptor 4, protein tyrosine kinase 2 beta, Bruton agammaglobulinemia tyrosine kinase, CDC-like kinase 1, lymphocyte-specific protein tyrosine kinase, GR feline sarcoma viral (v-fgr) oncogene homolog, anaplastic lymphoma kinase (Ki-1), fer (fms/fps related) protein kinase, EphB 1, fyn-related kinase, TTK protein kinase, Eph receptor B3, Eph receptor B4, fibroblast growth factor receptor 4.

Dependencies are inferred from the claim text.

Methods, reagents, kits and apparatus for protein function analysis

Life of this patent

Methods, reagents, kits and apparatus for protein function analysis

Life of this patent

Abstract

Claims132