HEPARINASE-III FROM FLAVOBACTERIUM-HEPARINUM - CLONING AND RECOMBINANT EXPRESSION IN ESCHERICHIA-COLI

Heparinase III (E.C. 4.2.2.8), formerly hepartinase I, produced by Fla vobacterium heparinum is an enzyme that specifically cleaves heparan s ulfate-rich regions of acidic polysaccharides. In this study, we repor t the cloning of the heparinase III gene using polymerase chain reacti on (PCR). Two degenerate oligonucleotides, based on amino acid sequenc es derived from tryptic peptides of purified heparinase III were used to generate a similar to 1100-bp probe by PCR amplification using Flav obacterium genomic DNA as the template. The PCR-derived probe was used to screen a Flavobacterium genomic DNA library in lambda ZAP II. The open reading frame of the heparinase III gene is 1980 bp in length, en coding a precursor protein of 75,950 Da; 10 of the tryptic peptides ma pped onto the open reading frame which corresponded to similar to 18% of the protein. Recombinant heparinase III was expressed in Escherichi a coli using the T7 polymerase pET expression system. This is the firs t report of the cloning and recombinant expression of an enzyme primar ily degrading heparan sulfate. (C) 1996 Academic Press, Inc.