ISOLATION AND EXPRESSION IN ESCHERICHIA-COLI OF HEPB AND HEPC, GENES-CODING FOR THE GLYCOSAMINOGLYCAN-DEGRADING ENZYMES HEPARINASE-II AND HEPARINASE-III, RESPECTIVELY, FROM FLAVOBACTERIUM-HEPARINUM

Upon induction with heparin, Flavobacterium heparinum synthesizes and secretes into its periplasmic space heparinase I (EC 4.2.2.7), heparin ase II, and heparinase III (heparitinase; EC 4.2.2.8), Heparinase I de grades heparin, and heparinase II degrades both heparin and heparan su lfate, while heparinase III degrades heparan sulfate predominantly, We isolated the genes encoding heparinases II and III (designated hepB a nd hepC, respectively), These genes are not contiguous with each other or with the heparinase I gene (designated hepA). hepB and hepC were f ound to contain open reading frames of 2,316 and 1,980 bp, respectivel y, Enzymatic removal of pyroglutamate groups permitted sequence analys is of the amino termini of both mature proteins, It was determined tha t the mature forms of heparinases II and III contain 746 and 635 amino acids, respectively, and have calculated molecular weights of 84,545 and 73,135, respectively, The preproteins have signal sequences consis ting of 26 and 25 amino acids, Truncated hepB and hepC genes were used to produce active, mature heparinases II and III in the cytoplasm of Escherichia coli, When these enzymes were expressed at 37 degrees C, m ost of each recombinant enzyme was insoluble, and most of the heparina se III protein was degraded, When the two enzymes were expressed at 25 degrees C, they were both present predominantly in a soluble, active form.