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AssigneeCarlsberg A/S
InventorsKlaus Breddam, Morten C. Kielland-Brandt, Uffe Hasbo Mortensen, Kjeld Ove Olesen, Henning Ralf Stennicke, Fred W. Wagner
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The invention provides customized proteases (i.e., mutant enzymes), methods of making customized proteases, as well as methods of using customized proteases. The customized proteases of the invention are derived from the known proteases. Altered transacylation reactions include the capability to perform transacylation reactions not substantially catalyzed by the known protease or the capability to perform transacylation reactions with improved yields, or both. The methods of the invention provide for customized proteases through site specific or random mutagenesis of the active site amino acids of the known proteases. The invention also provides for methods of using the customized proteases to prepare a preselected transacylation products. The preselected transacylation products produced can be modified by substitution at the N-or C-terminal with nucleophiles such as L-amino acids, D-amino acids, amino acid amides, and radioactive amino acids.
A customized carboxypeptidase derived from carboxypeptidase Y, the customized carboxypeptidase comprising an S1′ subsite modified by substitution, addition, or deletion of at least one amino acid residue, the customized carboxypeptidase capable of more effectively catalyzing transacylation of a preselected peptide substrate with a preselected nucleophile as compared with the carboxypeptidase Y.
A process for modifying a preselected peptide substrate by transacylation using a customized carboxypeptidase derived from carboxypeptidase Y, comprising:
The process of claim 30, wherein the preselected substrate comprises GRF (1-43)-Ala (SEQ ID NO:2) and the preselected nucleophile comprises leucine amide.
The process of claim 30, wherein the preselected substrate is GRF (1-43)-Ala (SEQ ID NO:2) and the preselected nucleophile comprises leucine amide.
Dependencies are inferred from the claim text.