National Research Council of Canada

Canadian Patents & Development Ltd.

Daniel Dignard

Howard Bussey

David Y Thomas

Daniel C Tessier

John J M Bergeron

Aleksandra Dmochowska

The present invention relates to a method for determining the effect of a test sample on UGGT activity. The method comprises the steps of: a) exposing an acceptor substrate of UGGT such as acid phosphatase to a labeled donor such as UDP- H-glucose in the presence of the test sample and UGGT; and b) detecting the amount of donor which was transferred to the acceptor substrate wherein a decrease of donor intake when compared to a control means that the test sample is a UGGT stimulator and a decrease means that the test sample is a UGGT inhibitor. The present invention also relates to an isolated mammalian cDNA which encodes for rat UGGT and to methods of producing mammalian UGGT using recombinant vectors.

Methods for screening UDP-glucose:glycoprotein glucosyltransferase (UGGT) activity and nucleic acid encoding for UGGT

This invention is concerned with the specific processing of secreted proteins in genetically modified yeast cells. The yeast KEX1 gene was cloned and the KEX1 product was shown to be a serine protease, evidently a carboxypeptidase B-like protease. A probable site of processing of polypeptides by the KEX1 gene product is at the C-terminus of the .alpha. subunit of the killer toxin, where the mature toxin subunit is followed in the precursor by a pair of basic amino acid residues. Processing likely involves an endoprotease cut following these basic residues, and their subsequent C-terminal trimming by a carboxypeptidase. Consistent with the KEX1 product being this carboxypeptidase is the finding that it is also involved in .alpha.-factor pheromone production. In wildtype yeast, KEX1 is not essential for .alpha.-factor production, as the final hormone repeat in the prepro .alpha.-hormone precursor does not need C-terminal processing to form one copy of the active hormone. However, in a mutant strain where .alpha.-factor production requires carboxypeptidase B-like processing, pheromone production is KEX1 dependent. Besides the processing of yeasts' naturally secreted proteins, of which .alpha.-factor pheromone and K1 killer toxin are the best characterized examples, the products of KEX1 and KEX2 are required for the processing of some proteins and peptides of commercial importance for example hormones and neuropeptides. High level production of certain commercially important proteins and peptides appears to require the overproduction of the appropriate processing proteases. The cloning of KEX1 on a multicopy plasmid can provide for this overproduction.

Protease for specific processing of secreted proteins

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Montréal, Canada

Daniel Dignard