CLONING, SEQUENCING AND EXPRESSION OF A CDNA-ENCODING BOVINE PANCREATIC DEOXYRIBONUCLEASE-I IN ESCHERICHIA-COLI - PURIFICATION AND CHARACTERIZATION OF THE RECOMBINANT ENZYME

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 read ing frame which can be translated as a 282-amino acid polypeptide, inc luding a hydrophobic signal peptide and the polypeptide of bp-DNase I. An expression plasmid was constructed by inserting into the vector pE T-15b, a cDNA fragment coding for bp-DNase I ligated with a hexanucleo tide coding for Met-Ala at the 5'-end. The plasmid was transformed int o E. coli strain DH5 alpha and the active recombinant bovine (rb-) DNa se I was produced after induction of protein synthesis. From the induc ed 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 a nd had the same specific activity as bp-DNase I. The NH2-terminus of r b-DNase I was Ala, not Met, and at position 19, corresponding to the c arbohydrate attachment site of bp-DNase I, Asn was not glycosylated. ( C) 1998 Elsevier Science B.V.