Heparinase III from Flavobacterium heparinum: cloning and recombinant expression in Escherichia coli.
Heparinase III (E.C. 4.2.2.8), formerly heparinase I, produced by Flavobacterium heparinum is an enzyme that specifically cleaves heparan sulfate-rich regions of acidic polysaccharides. In this study, we report the cloning of the heparinase III gene using polymerase chain reaction (PCR). Two degenerate oligonucleotides, based on amino acid sequences derived from tryptic peptides of purified heparinase III were used to generate a approximately 1100-bp probe by PCR amplification using Flavobacterium genomic DNA as the template. The PCR-derived probe was used to screen a Flavobacterium genomic DNA library in lambda ZAP II. The open reading frame of the heparinase III gene is 1980 bp in length, encoding a precursor protein of 75,950 Da; 10 of the tryptic peptides mapped onto the open reading frame which corresponded to approximately 18% of the protein. Recombinant heparinase III was expressed in Escherichia coli using the T7 polymerase pET expression system. This is the first report of the cloning and recombinant expression of an enzyme primarily degrading heparan sulfate.
Godavarti
R
R
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge 02139, USA.
Davis
M
M
Venkataraman
G
G
Cooney
C
C
Langer
R
R
Sasisekharan
R
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005417:Flavobacterium / Q000201:enzymology* / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D019763:Heparin Lyase; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010446:Peptide Fragments / Q000235:genetics; D016133:Polymerase Chain Reaction; D011133:Polysaccharide-Lyases / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification
D017931:DNA Primers; D004269:DNA, Bacterial; D010446:Peptide Fragments; D011994:Recombinant Proteins; D011133:Polysaccharide-Lyases; D019763:Heparin Lyase