Chicken deoxyribonuclease: purification, characterization, gene cloning and gene expression.
Chicken DNase was purified to apparent homogeneity from the pancreas extract. It showed two isoforms, A and B forms, on cation-exchange chromatography. On SDS-PAGE it was a 30-kDa protein. When analyzed on an electrospray-mass analyzer, form A showed a major mass peak of 30859, and form B, 30882. The enzyme was bound to concanavalin A, indicating its glycoprotein nature. The carbohydrate side chain could be removed by endoglycosidase F. Chicken DNase was activated by metal ions and for half-maximum activation, Mn2+ and Mg2+ required were 1 mM and 4 mM, respectively. The pH optimum was between 7 and 8 depending on the metal ions used. In the presence of Cu2+, it was almost completely inactivated by 0.1 M iodoacetate within 1 min. In the absence of Ca2+ at pH 8, chicken DNase resisted to the trypsin or beta-mercaptoethenol inactivation. When the purified enzyme was subjected to protein sequencing, approximately 93% of the sequence was established. Based on the amino acid sequence, the cDNA of chicken DNase was amplified, cloned and sequenced. The cDNA sequence consisted of 1079 nucleotides in which 67 were of the 5'-untranslated region and 166 of the 3' and, in the 5'-untranslated region, two types of sequences occurred. The polypeptide chain of 282 amino acids, translated from the open reading frame, was composed of the mature protein of 262 amino acids and a putative signal peptide of 20 amino acids. As compared with mammalian DNases, chicken DNase had an overall 58 +/- 61% sequence identity, one less potential N-glycosylation site, and one extra disulfide. The cDNA was cloned into the pET15b expression vector. When induced, active recombinant chicken DNase was expressed in Escherichia coli strain BL21(DE3)pLysS and was present in the insoluble fraction of cell lysates.
Hu
Chi-Chao
CC
Institute of Biochemistry, College of Medicine, National Taiwan University, Taipei, Taiwan.
Lu
Shao-Chun
SC
Cheng
Chao-Cheng
CC
Chen
Li-Hua
LH
Liao
Ta-Hsiu
TH
D000818:Animals; D001483:Base Sequence; D002645:Chickens / Q000201:enzymology*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis; D003851:Deoxyribonucleases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics*; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D018076:DNA, Complementary; D011994:Recombinant Proteins; D003851:Deoxyribonucleases