Methods, reagents, kits and apparatus for protein function analysis

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 plurality of nucleic acid constructs,
• b) simultaneously transcribing said nucleic acid constructs in different wells of a multi-well plate to generate RNAs,
• c) simultaneously translating said RNAs in said wells of said multi-well plate, using a cell-free system containing a tRNA precharged with a modified amino acid, to produce a plurality of amino acid sequences containing said modified amino acid, and
• d) detecting said post-translational modification of said plurality of amino acid sequences by a solid phase binding assay for the binding of said amino acid sequences to binding species specific for said post-translational modification.

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 plurality of nucleic acid constructs,
• (b) simultaneously transcribing said nucleic acid constructs in different wells of a multi-well plate to generate RNAs,
• (c) simultaneously translating said RNAs in said wells of said multi-well plate, using 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 at least one 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.

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.

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,
• (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.

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.

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 construct 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,
• (ii) capturing said binding species on a surface of a well of a multi-well plate via a second binding species and
• (iii) detecting said detectable species bound to said surface.

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.

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.

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.

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.

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-transitional modification, to produce an amino acid sequence containing said affinity tag and said post-translational modification,
• (d) immobilizing said amino acid sequence on a solid phase surface using binding species specific for said post-translational modification, and
• (e) detecting said amino acid sequence using a binding reagent specific for said affinity tag.

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
• iii) detecting the product of the enzyme activity.