Cloning and expression of heparinase I gene from Flavobacterium heparinum.
Heparinases, enzymes that cleave heparin and heparin sulfate, are implicated in physiological and pathological functions ranging from wound healing to tumor metastasis and are useful in deheparinization therapies. We report the cloning of the heparinase I (EC 4.2.2.7) gene from Flavobacterium heparinum using PCR. Two degenerate oligonucleotides, based on the amino acid sequences derived from tryptic peptides of purified heparinase, were used to generate a 600-bp probe by PCR amplification using Flavobacterium genomic DNA as the template. This probe was used to screen a Flavobacterium genomic DNA library in pUC18. The open reading frame of heparinase I is 1152 bp in length, encoding a precursor protein of 43.8 kDa. Eleven of the tryptic peptides (approximately 35% of the total amino acids) mapped onto the open reading frame. The amino acid sequence reveals a consensus heparin binding domain and a 21-residue leader peptide with a characteristic Ala-(Xaa)-Ala cleavage site. Recombinant heparinase was expressed in Escherichia coli as a soluble protein, using the T7 polymerase pET expression system. The recombinant heparinase cleavage of heparin was identical to that of native heparinase.
Sasisekharan
R
R
Division of Medical Sciences, Harvard Medical School, Boston, MA 02115.
Bulmer
M
M
Moremen
K W
KW
Cooney
C L
CL
Langer
R
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular / Q000379:methods; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005417:Flavobacterium / Q000201:enzymology* / Q000235:genetics*; D005798:Genes, Bacterial; D015698:Genomic Library; D019763:Heparin Lyase; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D009844:Oligosaccharides / Q000302:isolation & purification; D010446:Peptide Fragments / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods; D011133:Polysaccharide-Lyases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D004269:DNA, Bacterial; D009838:Oligodeoxyribonucleotides; D009844:Oligosaccharides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D011133:Polysaccharide-Lyases; D019763:Heparin Lyase