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AssigneeCangene Corporation
InventorsDaniel Bartfeld, Michael J. Butler, Dany Hadary, David L. Jenish, Timothy J. Krieger, Lawrence T. Malek, Gisela Soostmeyer, Eva Walczyk, Phyllis Krygsman, Sheila Garven
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A family of proteases endogenous to Streptomyces cells degrades heterologous proteins secreted from Streptomyces host cells. The previously unidentified proteases include (1) tripeptidyl aminopeptidase--Streptomyces ("Tap"), (2) a Streptomyces protease ("Ssp") which displayed significant amino acid sequence homology to Subtilisin BPN' and showed an ability to remove tripeptides from the amino termini of proteins and peptides, and (3) other proteases derived from Streptomyces which degraded certain substrates under certain conditions. Degradation was alleviated by selective inhibition of secreted proteases or by using hosts with impaired capabilities to produce proteases. An irreversible inhibitor was designed based upon the mechanism and substrate specificity of the target protease. Hosts secreting high amounts of proteases were selected. Impaired hosts were produced by deleting or altering the nucleotide sequence for the proteases.
An isolated and purified tripeptidyl aminopeptidase of Streptomyces, wherein (A) said aminopeptidase is endogenous to Streptomyces, (B) said aminopeptidase cleaves an amino terminal tripeptide from a polypeptide, and (C) said terminal peptide has the sequence:
Isolated and purified SlpD protein that has an approximate molecular weight of 55 kDa and comprises the amino acid sequence of SEQ ID NO:8 from amino acid 17 to amino acid 492.
An isolated and purified protease, wherein said protease has the amino acid sequence of SEQ ID NO:6.
Isolated and purified SlpE protein that has an approximate molecular weight of 56 kDa and comprises the amino acid sequence of SEQ ID NO:6 from amino acid 22 to amino acid 512.
A polypeptide comprising, in the order of translation, a signal peptide, a propeptide comprising at least one tripeptide, and the amino acid sequence of a mature protein, wherein said amino acid sequence has a structural constraint that would inhibit cleavage of said signal peptide by a signal peptidase in the absence of said propeptide, and wherein said tripeptide has the sequence:
A method for improving the secretion of a mature protein by a transformed host cell, comprising the steps of:
Dependencies are inferred from the claim text.