Ubiquitin-specific proteases

5,683,904

08/487,203

June 07, 1995

The disclosure relates to a generic class of ubiquitin-specific proteases which specifically cleave at the C-terminus of the ubiquitin moiety in a ubiquitin fusion protein irrespective of the size of the ubiquitin fusion protein. More specifically, the disclosure relates to ubiquitin-specific proteases of this class which have been isolated from a cell. The disclosure also relates to isolated DNA sequences encoding the proteases of this class.

Baker, Rohan T. (Garran, AUX); Tobias, John W. (Cambridge, MA); Varshavsky, Alexander (Boston, MA)

Massachusetts Institute of Technology (Cambridge, MA)

We claim

1. An isolated DNA encoding a ubiquitin-specific protease which specifically cleaves a ubiquitin fusion protein having a molecular weight of about 120 kilodaltons, the specific cleavage taking place in vitro between the C-terminal residue of ubiquitin and the N-terminal residue of the protein or peptide, the fusion protein being encoded by the DNA represented in Sequence ID Number 1.

2. An isolated DNA of claim 1 which is characterized by the ability to hybridize specifically with the DNA represented in Sequence I.D. Number 3 under stringent hybridization conditions.

3. An isolated DNA of claim 1 which is characterized by the ability to hybridize specifically with the DNA represented in Sequence I.D. Number 5 under stringent hybridization conditions.

4. An isolated DNA encoding a ubiquitin-specific protease which specifically cleaves a ubiquitin fusion protein having a molecular weight of about 120 kilodaltons, the specific cleavage taking place in a prokaryotic cell between the C-terminal residue of ubiquitin and the N-terminal residue of the protein or peptide, the fusion protein being encoded by the DNA represented in Sequence I.D. Number 1.

5. An isolated DNA of claim 4 which is characterized by the ability to hybridize specifically with the DNA represented in Sequence I.D. Number 3 under stringent hybridization conditions.

6. An isolated DNA of claim 4 which is characterized by the ability to hybridize specifically with the DNA represented in Sequence I.D. Number 5 under stringent hybridization conditions.

7. An isolated DNA of claim 4 which is characterized by the ability to hybridize specifically with the DNA represented in Sequence I.D. Number 7 under stringent hybridization conditions.

8. A cell transformed with

a) a first DNA expression construct encoding a biologically active ubiquitin-specific protease comprising a DNA sequence selected from the group consisting of Sequence I.D. Number 3, Sequence I.D. Number 5 and Sequence I.D. Number 7, or a portion of these sequences, said portion encoding said protease, in expressible form; and

b) a second DNA expression construct encoding ubiquitin joined to a DNA sequence encoding a protein or polypeptide of interest having a predetermined amino acid residue at its amino terminus, the ubiquitin being proteolytically cleavable by a ubiquitin-specific endoprotease at the junction with the amino-terminus of the protein or polypeptide of interest such that cleavage results in the exposure of the predetermined amino-terminal residue of the protein or polypeptide of interest.

9. A cell of claim 8 which is a prokaryotic cell.

10. A cell of claim 8 which is E. coli.

4352/523; 435/212; 435/219; 43525/233; 435/243; 4353/201; 435/6; 536/232; 536/234

C12N 120; C12N 950; C12N 948; C12N 100

5108919 April 1992 Liu et al.
5132213 July 1992 Bachmair et al.
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