Expression in Escherichia coli, purification and characterization of heparinase I from Flavobacterium heparinum.

ABSTRACT: The use of heparin for extracorporeal therapies has been problematical due to haemorrhagic complications; as a consequence, heparinase I from Flavobacterium heparinum is used for the determination of plasma heparin and for elimination of heparin from circulation. Here we report the expression of recombinant heparinase I in Escherichia coli, purification to homogeneity and characterization of the purified enzyme. Heparinase I was expressed with an N-terminal histidine tag. The enzyme was insoluble and inactive, but could be refolded, and was purified to homogeneity by nickel-chelate chromatography. The cumulative yield was 43%, and the recovery of purified heparinase I was 14.4 mg/l of culture. The N-terminal sequence and the molecular mass as analysed by matrix-assisted laser desorption MS were consistent with predictions from the heparinase I gene structure. The reverse-phase HPLC profile of the tryptic digest, the Michaelis-Menten constant Km (47 micrograms/ml) and the specific activity (117 units/mg) of purified recombinant heparinase I were similar to those of the native enzyme. Degradation of heparin by heparinase I results in a characteristic product distribution, which is different from those obtained by degradation with heparinase II or III from F. heparinum. We developed a rapid anion-exchange HPLC method to separate the products of enzymic heparin degradation, using POROS perfusion chromatography media. Separation of characteristic di-, tetra- and hexa-saccharide products is performed in 10 min. These methods for the expression, purification and analysis of recombinant heparinase I may facilitate further development of heparinase I-based medical therapies as well as further investigation of the structures of heparin and heparan sulphate and their role in the extracellular matrix.

SUBMITTER: Ernst S 

PROVIDER: S-EPMC1217237 | biostudies-other | 1996 Apr

REPOSITORIES: biostudies-other