Cloning, sequencing and expression of a cDNA encoding bovine pancreatic deoxyribonuclease I in Escherichia coli: Purification and characterization of the recombinant enzyme
Journal
Gene
Journal Volume
206
Journal Issue
2
Pages
181-184
Date Issued
1998
Author(s)
Chen C.-Y.
SHAO-CHUN LU
Liao T.-H.
Abstract
The bovine pancreatic (bp-) DNase I gene has been cloned from bp-cDNA and expressed in E. coli. A polynucleotide sequence of 1295 base pairs was deduced from clones of the cDNA. The sequence showed an open reading frame which can be translated as a 282-amino acid polypeptide, including a hydrophobic signal peptide and the polypeptide of bp-DNase I. An expression plasmid was constructed by inserting into the vector pET-15b, a cDNA fragment coding for bp-DNase I ligated with a hexanucleotide coding for Met-Ala at the 5'-end. The plasmid was transformed into E. coli strain DH5alpha and the active recombinant bovine (rb-) DNase I was produced after induction of protein synthesis. From the induced culture medium, rb-DNase I was purified by chromatography on a Mono Q column. The purified rb-DNase I showed a molecular mass of 29 kDa and had the same specific activity as bp-DNase I. The NH2-terminus of rb-DNase I was Ala, not Met, and at position 19, corresponding to the carbohydrate attachment site of bp-DNase I, Asn was not glycosylated.
Publisher
Elsevier
Type
journal article