Split- ubiquitin based reporter systems and methods of their use

A pair of fusion proteins consisting of a first fusion protein comprising segments P1, Cub-X, and RM, in an order wherein Cub-X is closer to the N-terminus of the first fusion protein than RM, and a second fusion protein comprising segments Nux and P2, wherein:

• P1 or P2 or both is a transcription factor;
• Nux is the amino-terminal subdomain of a wild-type ubiquitin or a reduced-associating mutant ubiquitin amino-terminal subdomain;
• Cub is the carboxy-terminal subdomain of a wild-type ubiquitin;
• X is an amino acid other than methionine;
• RM is an reporter moiety, and,
• wherein the binding that occurs between P1 and P2 results in reassociation of Nux and Cub, thereby permitting ubiquitin-specific protease cleavage between Cub and X.

A pair of fusion proteins consisting of a first fusion protein comprising segments P1, Cub-X, and RM, in an order wherein Cub-X is closer to the N-terminus of the first fusion protein than RM, and a second fusion protein comprising segments Nux and P2, wherein:

• P1 or P2 or both is a membrane-associated protein, and P2 may be the same or different from P1;
• Nux is the amino-terminal subdomain of a wild-type ubiquitin or a reduced-associating mutant ubiquitin amino-terminal subdomain;
• Cub is the carboxy-terminal subdomain of a wild-type ubiquitin;
• X is an amino acid;
• RM is an enzymatically active reporter moiety, and,
• wherein the binding that occurs between P1 and P2 results in reassociation of Nux and Cub, thereby permitting ubiquitin-specific protease cleavage between Cub and X.

A pair of fusion proteins consisting of a first fusion protein comprising segments P1, Cub-X, and RM, in an order wherein Cub-X is closer to the N-terminus of the first fusion protein than RM, and a second fusion protein comprising segments Nux and P2, wherein:

• P1 or P2 or both is transcription factor;
• Nux is the amino-terminal subdomain of a wild-type ubiquitin or a reduced-associating mutant ubiquitin amino-terminal subdomain;
• Cub is the carboxy-terminal subdomain of a wild-type ubiquitin;
• X is an amino acid;
• RM is an enzymatically active reporter moiety, and,
• wherein the binding that occurs between P1 and P2 results in reassociation of Nux and Cub, thereby permitting ubiquitin-specific protease cleavage between Cub and X.

One or more nucleic acids that encodes or that together encode a first fusion protein comprising segments P1, Cub-X, and RM, in an order wherein Cub-X is closer to the N-terminus of the first fusion protein than RM, and a second fusion protein comprising segments Nux and P2, wherein:

• P1 or P2 or both is a membrane-associated protein, and P2 may be the same or different from P1;
• Nux is the amino-terminal subdomain of a wild-type ubiquitin or a reduced-associating mutant ubiquitin amino-terminal subdomain;
• Cub is the carboxy-terminal subdomain of a wild-type ubiquitin;
• X is an amino acid other than methionine;
• RM is a reporter moiety, and,
• wherein the binding that occurs between P1 and P2 results in reassociation of Nux and Cub, thereby permitting ubiquitin-specific protease cleavage between Cub and X.

One or more nucleic acids that encodes or that together encode a first fusion protein comprising segments P1, Cub-X, and RM, in an order wherein Cub-X is closer to the N-terminus of the first fusion protein than RM, and a second fusion protein comprising segments Nux and P2, wherein:

• P1 or P2 or both is a membrane-associated protein, and P2 may be the same or different from P1;
• Nux is the amino-terminal subdomain of a wild-type ubiquitin or a reduced-associating mutant ubiquitin amino-terminal subdomain;
• Cub is the carboxy-terminal subdomain of a wild-type ubiquitin;
• X is an amino acid;
• RM is an enzymatically active reporter moiety, and,
• wherein the binding that occurs between P1 and P2 results in reassociation of Nux and Cub, thereby permitting ubiquitin-specific protease cleavage between Cub and X.

One or more nucleic acids that encodes or that together encode a first fusion protein comprising segments P1, Cub-X, and RM, in an order wherein Cub-X is closer to the N-terminus of the first fusion protein than RM, and a second fusion protein comprising segments Nux and P2, wherein:

• P1 or P2 or both is a transcription factor;
• Nux is the amino-terminal subdomain of a wild-type ubiquitin or a reduced-associating mutant ubiquitin amino-terminal subdomain;
• Cub is the carboxy-terminal subdomain of a wild-type ubiquitin;
• X is an amino acid other than methionine;
• RM is a reporter moiety, and,
• wherein the binding that occurs between P1 and P2 results in reassociation of Nux and Cub, thereby permitting ubiquitin-specific protease cleavage between Cub and X.

One or more nucleic acids that encodes or that together encode a first fusion protein comprising segments P1, Cub-X, and RM, in an order wherein Cub-X is closer to the N-terminus of the first fusion protein than RM, and a second fusion protein comprising segments Nux and P2, wherein:

• P1 or P2 or both is a transcription factor;
• Nux is the amino-terminal subdomain of a wild-type ubiquitin or a reduced-associating mutant ubiquitin amino-terminal subdomain;
• Cub is the carboxy-terminal subdomain of a wild-type ubiquitin;
• X is an amino acid;
• RM is an enzymatically active reporter moiety, and,
• wherein the binding that occurs between P1 and P2 results in reassociation of Nux and Cub, thereby permitting ubiquitin-specific protease cleavage between Cub and X.

A method of characterizing the sequence of a protein that binds a target protein comprising the steps of:

• expressing a first and a second nucleic acid in a ubiquitin-specific protease expressing cell, which first nucleic acid encodes a target fusion protein comprising segments P1, Cub-X, and RM, in an order wherein Cub-X is closer to the N-terminus of the target fusion protein than RM, wherein P1 is the target protein, Cub is the carboxy-terminal subdomain of a wild-type ubiquitin, X is an amino acid selected from the group consisting of arg, lys, phe, leu, trp, his, asp, asn, tyr, ile, glu, cys and gln, and RM is an enzymatically active reporter moiety,
• which second nucleic acid encodes a candidate fusion protein comprising segments P2 and Nux, wherein the second nucleic acid is a member of a library containing multiple different nucleic acids differing in the P2 segments they encode, P2 is a candidate segment and Nux is the amino-terminal subdomain of a wild-type ubiquitin or a reduced-associating mutant ubiquitin amino-terminal subdomain;
• recovering a clone of the cell expressing the first and second nucleic acid under conditions wherein a cell is selectable only in the absence of the enzymatic activity of RM;
• and,
• characterizing the second nucleic acid encoding P2.

A method of characterizing the sequence of a protein that binds a target protein comprising the steps of:

• expressing a first and a second nucleic acid in a ubiquitin-specific protease expressing cell, which first nucleic acid encodes a target fusion protein comprising segments P1, Cub-X, and RM, in an order wherein Cub-X is closer to the N-terminus of the target fusion protein than RM, wherein P1 is the target protein, Cub is the carboxy-terminal subdomain of a wild-type ubiquitin, X is an amino acid selected from the group consisting of arg, lys, phe, leu, trp, his, asp, asn, tyr, ile, glu, cys and gln, and RM is an active reporter moiety, which second nucleic acid encodes a candidate fusion protein comprising segments P2 and Nux, wherein the second nucleic acid is a member of a library containing multiple different nucleic acids differing in the P2 segments they encode, P2 is a candidate segment and Nux is the amino-terminal subdomain of a wild-type ubiquitin or a reduced-associating mutant ubiquitin amino-terminal subdomain;
• recovering a clone of the cell expressing the first and second nucleic acid under conditions wherein a cell is selectable only in the absence of an activity of RM;
• and,
• characterizing the second nucleic acid encoding P2.

A kit for characterizing the sequence of a polypeptide that binds a target protein, which comprises:

• a first nucleic acid encoding a target fusion protein comprising a cloning site suitable for the insertion of a nucleic acid encoding a target protein sequence, segments Cub-X, and RM, in an order wherein Cub-X is closer to the N-terminus of the target fusion protein than RM, wherein Cub is the carboxy-terminal subdomain of a wild-type ubiquitin, X is an amino acid selected from the group consisting of arg, lys, phe, leu, trp, his, asp, asn, tyr, ile, glu, cys and gln, and RM is an active reporter moiety, which activity allows for selection, whereby a fusion protein comprising the target protein sequence, Cub-X and RM can be expressed;
• a second nucleic acid comprising an Nux segment encoding the amino-terminal subdomain of a wild-type ubiquitin or a reduced-associating mutant ubiquitin amino-terminal subdomain and a cloning site suitable for the insertion of a nucleic acid encoding a polypeptide sequence whereby a fusion protein comprising Nux and the polypeptide sequence can be expressed;
• and,
• instructions indicating that a nucleic acid encoding a defined target protein sequence is to be inserted into the first nucleic acid and members of a library of nucleic acids encoding candidate polypeptides are to be inserted into the second nucleic acid, in order to characterize a polypeptide that binds to the target protein.