High yield, purity and activity of soluble recombinant Bacteroides thetaiotaomicron GST-heparinase I from Escherichia coli.

Heparinase I from Flavobacterium heparinum, a source of diverse polysaccharidases, suffers from low yields, insufficient purity for structural studies and insolubility when expressed as a recombinant product in Escherichia coli that is devoid of glycosaminoglycan polysaccharidases. In this study, cDNA coding for the orthologue of F. heparinum heparinase I was constructed from genomic information from the mammalian gut symbiont Bacteroides thetaiotaomicron and expressed in E. coli as a fusion protein with GST at the N-terminus. This resulted in high yield (30 mg/g dry bacteria) of soluble product and facilitated one-step affinity purification to homogeneity. Purified heparinase I bearing the GST fusion exhibited a K(m) of 2.3 microM and V(max) of 42.7 micromol/min with a specific activity of 164 U/mg with heparin (average 12,000 Da) as substrate. The results indicate a 2-fold improvement in yield, specific activity and affinity for heparin as substrate over previous reports. The data suggest that the heparinase I from the gut symbiont exhibits a higher intrinsic affinity for heparin than that from F. heparinum. The purified GST fusion enzyme exhibited a requirement for Ca(2+) and a pH optimum between 6.7 and 7.3 that was similar to the enzyme freed of the N-terminal GST portion. Our study revealed that catalytic activity of heparinase I requires a reducing environment. The GST facilitated immobilization of heparinase I in solid phase either for clinical purposes or for structural studies in absence of interference by contaminating polysaccharidases.