Cangene Corporation

Bio-technology General Corporation

Migenix, Inc.

Daniel Bartfeld

Dany Hadary

Lawrence T Malek

Michael J Butler

Timothy J Krieger

Gisela Soostmeyer

David L Jenish

Phyllis Krygsman

Eva Walczyk

Sheila Garven

Meir Fischer

Benjamin Greenman

Ruth Lieshitz

Jàn Burian

Ruth Lifshitz

Eva Walcyzk

Endogenously produced cationic antimicrobial peptides are ubiquitous components of host defenses in mammals, birds, amphibia, insects, and plants. Cationic peptides are also effective when administered as therapeutic agents. A practical drawback in cationic peptide therapy, however, is the cost of producing the agents. The methods described herein provide a means to efficiently produce cationic peptides from recombinant host cells. These recombinantly-produced cationic peptides can be rapidly purified from host cell proteins using anion exchange chromatography.

Efficient methods for producing anti-microbial cationic peptides in host cells

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

A family of proteases endogenous to Streptomyces cells degrade 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 tripeprides 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 improved streptomyces strains for expression

Applicants describe methods for purifying human ApoE or analog thereof from recombinant bacterial cells with minimal protein aggregation and degradation during the purification process. The invention involves the addition of neutralized fatty acids to the medium during cell disruption and the use of a non-ionic detergent during the purification process. Additionally applicants describe a method for increasing the production of ApoE or analog thereof in a bacterial host by adding to the culture medium neutralized fatty acids, fatty acid precursors, triglycerides, triglyceride precursors or acetate. Pharmaceutical and diagnostic uses of the ApoE analog are described.

Method for production and purification or recombinant Apolipoprotein E

The subject invention provides a method for recovering a solution containing purified, enzymatically active Cu-Zn superoxide dismutase or a polypeptide analog thereof having substantially the same amino acid sequence as, and the bioloical activity of, naturally-occurring Cu-Zn superoxide dismutase from a composition which comprises cells containing Cu-Zn superoxide dismutase or a polypeptide analog thereof. The invention also provides a method of increasing the yield of recovered solutions having an increased concentration of b isoform of an enzymatically-active polypeptide analog of Cu-Zn superoxide dismutase from a composition which comprises cells containing a, b and c isoforms of the polypeptide analog.

Method for purification of recombinant copper/zinc (CU-ZN) superoxide

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North York, Canada

Daniel Bartfeld