Protease for specific processing of secreted proteins

Details Protease for specific processing of secreted proteins Protease for specific processing of secreted proteins

1987-05-29

1990-05-29

Wiseman, Thomas G.

Chemistry: molecular biology and microbiology

Treatment of micro-organisms or enzymes with electrical or...

Modification of viruses

4351721, 435255, 435256, 435940, 435942, 435320, 435 691, 530303, 935 13, 935 28, 935 69, 536 27, C12N 1500, C12N 100, C12P 2102

Patent

active

049295535

ABSTRACT:
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.

REFERENCES:
patent: 4546082 (1985-10-01), Kurjan
Julius et al. (1984), Cell, 37:1075-1089.
Julius et al. (1983), Cell, 32:839-852.
Bussey et al. (1983), Mol Cell Biol, 3:1362-1370.
Kramer et al. (1986), Science, 231:4745.
Kurjan, J. (1985), Mol Cell Biol, 5:787-796.
Fricker et al. (1986), Nature, 323:461-464.
Thim et al. (1986), PNAS, 83:6766-6770.

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