Cloning, sequencing, and expression of the gene from bacillus circulans that codes for a heparinase that degrades both heparin and heparan sulfate.
The gene, designated hep, coding for a heparinase that degrades both heparin and heparan sulfate, was cloned from Bacillus circulans HpT298. Nucleotide sequence analysis showed that the open reading frame of the hep gene consists of 3,150 bp, encoding a precursor protein of 1,050 amino acids with a molecular mass of 116.5 kDa. A homology search found that the deduced amino acid sequence has partial similarity with enzymes belonging to the family of acidic polysaccharide lyases that degrade chondroitin sulfate and hyaluronic acid. Recombinant mature heparinase (111.2 kDa) was produced by the addition of IPTG from Escherichia coli harboring pETHEP with an open reading frame of the mature hep gene and was purified to homogeneity by SDS-polyacrylamide gel electrophoresis. Analyses of substrate specificity and degraded disaccharides indicated that the recombinant enzyme acts on both heparin and HS, as does heparinase purified from the wild-type strain.
Yoshida
Eiichi
E
Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Japan.
Arakawa
Shinji
S
Matsunaga
Taizo
T
Toriumi
Shigeki
S
Tokuyama
Shinji
S
Morikawa
Kiyoshi
K
Tahara
Yasutaka
Y
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004187:Disaccharides / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial / Q000235:genetics; D006493:Heparin / Q000378:metabolism*; D019763:Heparin Lyase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D006497:Heparan Sulfate / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011133:Polysaccharide-Lyases / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
D004187:Disaccharides; D006493:Heparin; D019763:Heparin Lyase; D006497:Heparan Sulfate; D011133:Polysaccharide-Lyases; D011994:Recombinant Proteins