title: 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.
authors.affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge 02139, USA.
authors.forename: S
authors.initials: S
authors.lastname: Ernst
authors.forename: G
authors.initials: G
authors.lastname: Venkataraman
authors.forename: S
authors.initials: S
authors.lastname: Winkler
authors.forename: R
authors.initials: R
authors.lastname: Godavarti
authors.forename: R
authors.initials: R
authors.lastname: Langer
authors.forename: C L
authors.initials: CL
authors.lastname: Cooney
authors.forename: R
authors.initials: R
authors.lastname: Sasisekharan
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005417:Flavobacterium / Q000201:enzymology* / Q000235:genetics*; D015870:Gene Expression; D006493:Heparin / Q000032:analysis; D006494:Heparin Antagonists / Q000627:therapeutic use; D019763:Heparin Lyase; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011133:Polysaccharide-Lyases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D006494:Heparin Antagonists; D011994:Recombinant Proteins; D006493:Heparin; D011133:Polysaccharide-Lyases; D019763:Heparin Lyase