Methods of generating desired amino-terminal residues in proteins
AssigneeMassachusetts Institute of Technology
InventorsAndreas Bachmair, Daniel Finley, Alexander Varshavsky
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Abstract
A method of designing or modifying protein structure at the protein or genetic level to produce specified amino-termini in vivo are described. The method can be 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.
Claims8
A method for regulating the metabolic stability of a protein or peptide of interest which is subject to the N-end rule of protein degradation, comprising producing, in a eukaryotic cell, a heterologous ubiquitin fusion protein comprising the protein or polypeptide of interest fused at the amino terminal amino acid residue to the carboxyl-terminal residue of ubiquitin, the fusion protein being cleaved in the cell by a ubiquitin-specific protease at junction of the amino terminal amino acid residue of the protein or peptide of interest and ubiquitin to produce the protein or peptide or interest having an amino-terminal residue selected from the group consisting of the destabilizing class and the stabilizing class of amino acid residues.
A method for metabolically destabilizing a protein or peptide of interest which is subject to the N-end rule of protein degradation, comprising producing, in a eukaryotic cell, a heterologous ubiquitin fusion protein comprising the protein or polypeptide of interest fused at the amino terminal amino acid residue to the carboxyl-terminal residue of ubiquitin, the fusion protein being cleaved in the cell by a ubiquitin-specific protease at junction of the amino terminal amino acid residue of the protein or peptide of interest and ubiquitin to produce the protein or peptide of interest having an amino-terminal residue selected from the group consisting of the residues aspartic acid, histidine, tryptophan, alanine, serine, threonine, isoleucine, glutamic acid, tyrosine, glutamine, phenylalanine, leucine, asparagine, lysine and arginine.
A method for producing a metabolically unstable protein or peptide, comprising producing, in a eukaryotic cell, a heterologous fusion protein comprising a ubiquitin-proline-protein or a ubiquitin-proline-peptide fusion, the fusion protein being cleaved in the cell by a ubiquitin-specific protease at the junction of ubiquitin and proline to produce the proline-protein or proline-peptide species which is metabolically unstable.
A DNA construct encoding a ubiquitin fusion protein comprising a DNA sequence encoding ubiquitin joined 5' to a DNA sequence encoding a protein or peptide, the protein or peptide having a predetermined amino acid residue of the destabilizing class at its amino terminus.
Dependencies are inferred from the claim text.