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.
Luo
Yongde
Y
Center for Cancer and Stem Cell Biology, Texas A&M University, Institute of Biosciences and Technology (IBT), The Texas A&M Health Science Center, 2121 W. Holcombe Blvd., Houston, TX 77030-3303, USA.
Huang
Xinqiang
X
McKeehan
Wallace L
WL
D000595:Amino Acid Sequence; D000925:Anticoagulants / Q000378:metabolism / Q000494:pharmacology; D001439:Bacteroides / Q000201:enzymology*; D002384:Catalysis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005417:Flavobacterium / Q000201:enzymology / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics; D006493:Heparin / Q000378:metabolism / Q000494:pharmacology; D019763:Heparin Lyase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D012995:Solubility; D013379:Substrate Specificity
D000925:Anticoagulants; D011993:Recombinant Fusion Proteins; D006493:Heparin; D005982:Glutathione Transferase; D019763:Heparin Lyase