Cangene Corporation

Sheila Garven

Lawrence T Malek

Daniel Bartfeld

Dany Hadary

Gisela Soostmeyer

David L Jenish

Michael J Butler

Timothy J Krieger

Eva Walczyk

Phyllis Krygsman

An aminopeptidase inhibitor is used when expressing heterologous protein in a bacterial host, such as Streptomyces. Use of such an inhibitor inhibits degradation of the heterologous protein by aminopeptidases. Inhibitors are designed based upon the mechanism and substrate specificity of the target protease and expressed protein.

Method for expression of proteins in bacterial host cells

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.

Streptomyces proteases and methods for improved secretion of

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Oakville, Canada

Sheila Garven