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1. (WO1989012692) LARGE SCALE METHOD FOR PURIFICATION OF HIGH PURITY HEPARINASE

Pub. No.:    WO/1989/012692    International Application No.:    PCT/US1989/002434
Publication Date: Dec 28, 1989 International Filing Date: Jun 2, 1989
IPC: C07K 16/40
C12N 15/00
C12N 9/88
Applicants: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
Inventors: ZIMMERMAN, Joseph, J.
COONEY, Charles, L.
Title: LARGE SCALE METHOD FOR PURIFICATION OF HIGH PURITY HEPARINASE
Abstract:
The present invention is an improved process for purification of active heparinase and heparinase like enzymes from Gram negative organisms, in particular, Flavobacterium heparinum. The primary advantage of the process is the fact that is allows large scale processing and high yield of heparinase. The heparinase is released from the pariplasmic space of the organism by osmotic shock treatment, first into an osmotically stabilized medium, secondly into a non-stabilized medium having a pH of approximately pH 6.0 and 8.6 with subsequent release into a second non-stabilized medium containing approximately 0.15 M sodium chloride, followed by fractionation by cation exchange chromatography, and, optionally, electrophoresis or gel filtration chromatography. Two proteins having heparinase activity have been isolated, one having a molecular weight of approximately 42,000 Daltons and the other having a molecular weight of 65,000 to 75,000 Daltons. Also described is the construction of a library for screening for the genes encoding the proteins having heparinase activity and two assays for detecting organisms producing heparinase, either F. heparinum or genetically engineered organisms.