Methods for generating catalytic proteins
OwnerMarkus Kurz, Peter Lohseinventors
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Abstract
Disclosed herein are novel methods for the generation and identification of catalytic and autoproteolytic proteins using nucleic acid-protein fusion approaches.
Claims40
A method for identifying a nucleic acid molecule which encodes a catalytic protein, said method comprising the steps of:
- a) providing a candidate catalytic protein fusion molecule, comprising a candidate catalytic protein linked to both its nucleic acid coding sequence and a substrate;
- and
- b) determining whether said candidate catalytic protein catalyzes a reaction of said substrate by assaying for an alteration in molecular size, charge, or conformation of said fusion molecule, relative to an unreacted fusion molecule, thereby identifying a nucleic acid molecule which encodes a catalytic protein.
A method for identifying a nucleic acid molecule which encodes a catalytic protein, said method comprising the steps of:
- a) providing a candidate catalytic protein fusion molecule, comprising a candidate catalytic protein linked to both its nucleic acid coding sequence and a substrate;
- b) allowing said candidate catalytic protein to catalyze a reaction of said substrate in solution;
- c) contacting the product of step (b) with a capture molecule that has specificity for and binds a reacted fusion molecule, but not an unreacted fusion molecule, said capture molecule being immobilized on a solid support;
- and
- d) detecting said reacted fusion molecule in association with said solid support,
- thereby identifying a nucleic acid molecule which encodes a catalytic protein.
The method of claim 6, wherein, as a result of said reaction, said substrate is covalently bonded to an affinity tag and said capture molecule binds said affinity tag but does not bind an unreacted fusion molecule.
A method for identifying a nucleic acid molecule which encodes a catalytic protein, said method comprising the steps of:
- a) providing a candidate catalytic protein fusion molecule, comprising a candidate catalytic protein linked to both its nucleic acid coding sequence and a substrate, said substrate being covalently bonded to an affinity tag;
- b) allowing said candidate catalytic protein to catalyze a reaction of said substrate in solution;
- c) contacting the product of step (b) with a capture molecule that is specific for said affinity tag, said capture molecule being immobilized on a solid support;
- and
- d) determining whether said fusion molecule is bound to said solid support, wherein the determination that a fusion molecule is not bound to said solid support identifies a nucleic acid molecule which encodes a catalytic protein.
A method for identifying a nucleic acid molecule which encodes a catalytic protein, said method comprising the steps of:
- a) providing a candidate catalytic protein fusion molecule, comprising a candidate catalytic protein linked to both its nucleic acid coding sequence and a substrate;
- b) allowing said candidate catalytic protein to catalyze a reaction of said substrate in solution in the presence of an affinity tag, said reaction resulting in the covalent attachment of said affinity tag to said fusion molecule;
- c) immunoprecipitating the product of step (b) with an antibody that is specific for said affinity tag;
- and
- d) detecting said immunoprecipitation complex, thereby identifying said fusion molecule as having a nucleic acid molecule which encodes a catalytic protein.
A method for identifying a nucleic acid molecule which encodes an autoproteolytic protein, said method comprising the steps of:
- a) providing a candidate autoproteolytic protein fusion molecule, comprising a candidate autoproteolytic protein linked to its nucleic acid coding sequence;
- and
- b) determining whether said candidate autoproteolytic protein catalyzes a self-reaction by assaying for an alteration in molecular size, charge, or conformation of said fusion molecule, relative to an unreacted fusion molecule, thereby identifying a nucleic acid molecule which encodes an autoproteolytic protein.
A method for identifying a nucleic acid molecule which encodes an autoproteolytic protein, said method comprising the steps of:
- a) providing a candidate autoproteolytic protein fusion molecule, comprising a candidate autoproteolytic protein linked to its nucleic acid coding sequence;
- b) allowing said candidate autoproteolytic protein to self-react;
- c) contacting the product of step (b) with a capture molecule that has specificity for and binds a self-reacted fusion molecule, but not an unreacted fusion molecule, said capture molecule being immobilized on a solid support;
- and
- d) detecting said self-reacted fusion molecule in association with said solid support, thereby identifying a nucleic acid molecule which encodes an autoproteolytic protein.
A method for identifying a nucleic acid molecule which encodes an autoproteolytic protein, said method comprising the steps of:
- a) providing a candidate autoproteolytic protein fusion molecule, comprising a candidate autoproteolytic protein linked to its nucleic acid coding sequence, said protein being covalently bonded to an affinity tag;
- b) allowing said candidate autoproteolytic protein to self-react in solution;
- c) contacting the product of step (b) with a capture molecule that is specific for said affinity tag, said capture molecule being immobilized on a solid support;
- and
- d) determining whether said fusion molecule is bound to said solid support, wherein the determination that a fusion molecule not bound to said solid support identifies a nucleic acid molecule which encodes an autoproteolytic protein.
A method for identifying a nucleic acid molecule which encodes an autoproteolytic protein, said method comprising the steps of:
- a) providing a candidate autoproteolytic protein fusion molecule, comprising a candidate autoproteolytic protein linked to its nucleic acid coding sequence;
- b) allowing said candidate autocatalytic protein to self-react in solution;
- c) immunoprecipitating the product of step (b) with an antibody that is specific for a reacted fusion molecule;
- and
- d) detecting said immunoprecipitation complex, thereby identifying said fusion molecule as having a nucleic acid molecule which encodes an autoproteolytic protein.
Dependencies are inferred from the claim text.