US5132213A
Expired / Lapsed
US
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Method for producing proteins and polypeptides using ubiquitin fusions
Assignee: Massachusetts Institute of Technology
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Filing: 1988-04-07
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Granted: 1992-07-21
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Expires: 2008-04-07
Abstract
Abstract A method of designing or modifying protein structure at the protein or genetic level to produce specified amino-termini in vivo is described. The method is used to alter the metabolic stability and other properties of the protein or, alternatively, to artificially generate authentic amino-termini in proteins produced through artificial means. The method is based upon the introduction of the use of artificial ubiquitin-protein fusions, and the discovery that the in vivo half-life of a protein is a function of the amino-terminal amino acid of the protein.
Inventors
- 👤 Andreas Bachmair
- 👤 Daniel Finley
- 👤 Alexander Varshavsky
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-04 20:58
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/0004; C12N9/0012; C12N9/0026; C12N9/0028; C07; C07K; C07K1/00; C07K1/107; C07K1/1072; C07K1/1075; C07K14/00; C12N15/00; C12N15/09; C12N15/11; C12N15/62; C12N15/63; C12N15/67; C12N9/14; C12N9/48; C12N9/50; C12N9/58; C12N9/60; C12N9/64; C12N9/6421; C12P; C12P21/00; C12P21/02; C07K2319/00; C07K2319/50; C07K2319/61; C07K2319/95; Y; Y10; Y10S; Y10S435/00; Y10S435/8215; Y10S435/911; Y10S435/94
Claims Analysis
Total Claims: 8
Claim 1
Independent Claim
90 words
A method of producing a protein or polypeptide having a predetermined amino-terminal amino acid residue, comprising expressing the protein or polypeptide as a heterologous fusion protein in a eukaryotic host cell wherein the amino terminus of the protein or polypeptide is fused to ubiquitin which is specifically proteolytically cleavable at the junction with the amino-terminal amino acid residue of the protein or polypeptide, said host cell containing a ubiquitin-specific protease which specifically cleaves the fusion protein at the junction with the amino-terminal amino acid residue of the protein or polypeptide.
Claim 2
Independent Claim
90 words
A method of producing a protein or polypeptide having a predetermined amino-terminal amino acid residue, comprising expressing the protein or polypeptide as a heterologous fusion protein in a eukaryotic host cell wherein the amino terminus of the protein or polypeptide is fused to ubiquitin which is specifically proteolytically cleavable at the junction with the amino-terminal amino acid residue of the protein or polypeptide, said host cell containing a ubiquitin-specific protease which specifically cleaves the fusion protein at the junction with the amino-terminal amino acid residue of the protein or polypeptide.
Claim 3
Independent Claim
136 words
A method of producing a protein having a predetermined amino-terminal amino acid residue, comprising: a. preparing a DNA construct encoding a heterologous ubiquitin fusion protein comprising: i. a gene encoding the protein, the gene having a sequence at its 5' end encoding the predetermined amino-terminal amino acid residue of the protein; and ii) DNA encoding ubiquitin linked to the 5' end of the gene encoding the protein, ubiquitin being specifically proteolytically cleavable at its junction with the amino terminus of the protein by a ubiquitin-specific endoprotease; and b. expressing the DNA construct in a eukaryotic host cell which contains a ubiquitin-specific protease which cleaves the heterologous fusion product encoded by the DNA construct at the junction of ubiquitin and the amino terminus of the protein to provide a protein having the predetermined amino-terminal amino acid residue.
Claim 4
Independent Claim
136 words
A method of producing a protein having a predetermined amino-terminal amino acid residue, comprising: a. preparing a DNA construct encoding a heterologous ubiquitin fusion protein comprising: i. a gene encoding the protein, the gene having a sequence at its 5' end encoding the predetermined amino-terminal amino acid residue of the protein; and ii) DNA encoding ubiquitin linked to the 5' end of the gene encoding the protein, ubiquitin being specifically proteolytically cleavable at its junction with the amino terminus of the protein by a ubiquitin-specific endoprotease; and b. expressing the DNA construct in a eukaryotic host cell which contains a ubiquitin-specific protease which cleaves the heterologous fusion product encoded by the DNA construct at the junction of ubiquitin and the amino terminus of the protein to provide a protein having the predetermined amino-terminal amino acid residue.
Claim 5
Dependent Claim
24 words
A method of claim 2, wherein the fusion protein is cleaved within the host cell to release the protein having the predetermined amino-terminal structure.
Claim 6
Dependent Claim
13 words
A method of claim 2, wherein the host cell is a yeast cell.
Claim 7
Dependent Claim
80 words
A gene construct encoding a heterologous fusion protein, the gene construct comprising a DNA sequence 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 or interest.
Claim 8
Dependent Claim
80 words
A gene construct encoding a heterologous fusion protein, the gene construct comprising a DNA sequence 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 or interest.
References Cited (Backward Citations)
US4667017A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1995-07-21 | 1996-01-20 | 1996-07-21 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1999-07-21 | 2000-01-20 | 2000-07-21 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2003-07-21 | 2004-01-20 | 2004-07-21 | $7700.00 | Window Closed / Lapsed |