Cloning, sequencing and expression of a cDNA encoding bovine pancreatic deoxyribonuclease I in Escherichia coli: purification and characterization of the recombinant enzyme

Chen, Ching-Ying; Lu, Shao-Chun; Liao, Ta–Hsiu

doi:10.1016/s0378-1119(97)00582-9

Cited by 23 publications

(14 citation statements)

References 13 publications

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“…TPCK‐Trypsin and bpDNase (code DP) was purchased from Worthington Biochemical Corporation. The purchased bpDNase was further purified on anion‐exchange chromatography (Chen et al 1998). NPPP, β‐mercaptoethanol, EGTA, OCPC, AMP, and horseradish peroxidaseconjugated secondary antibodies were purchased from Sigma.…”

Section: Methodsmentioning

confidence: 99%

The distinctive functions of the two structural calcium atoms in bovine pancreatic deoxyribonuclease

Chen

Liao

2002

Protein Science

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The two amino acid residues, Asp 99 and Asp 201, involved in the coordination of the two calcium atoms in the X-ray structure of bovine pancreatic (bp) DNase, were individually changed by site-directed mutagenesis. The two altered proteins, brDNase(D99A) and brDNase(D201A) were expressed in Escherichia coli and purified by anion exchange chromatography. Equilibrium dialysis showed that mutation destroyed one Ca 2+ -binding site each in brDNase(D99A) and brDNase(D201A). Compared with bpDNase, the Vmax value for brDNase(D99A) remained unchanged and that for brDNase(D201A) was decreased, whereas the K m values for the two variants were increased two-to threefold when the DNA hydrolytic hyperchromicity assay was used. Like bpDNase, brDNase(D99A) was able to make double scission on duplex DNA with Mg 2+ plus Ca 2+ and was effectively protected by Ca 2+ from the trypsin inactivation. But under the same conditions, brDNase(D201A) lost the double-scission ability and was not protected by Ca 2+ . Nevertheless, the two variant proteins retained the characteristics of the Ca 2+ -induced conformational changes and the Ca 2+ protection against the ␤-mercaptoethanol disruption of the essential disulfide bond, suggesting that other weaker Ca 2+ -binding sites not found in the X-ray structure were responsible for these properties. Therefore, the two structural calcium atoms are not for maintaining the overall conformation of the active DNase, as it has been indicated in the X-ray analysis, but rather play the role in the fine-tuning of the DNase activity.

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Section: Methodsmentioning

confidence: 99%

The distinctive functions of the two structural calcium atoms in bovine pancreatic deoxyribonuclease

Chen

Liao

2002

Protein Science

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“…The amino acid sequence of the polypeptide chain has been elucidated by chemical analysis [16]. A gene encoding bpDNase was originally chemically synthesized [17], and later another was cloned from the bp-cDNA [18] and they were expressed in E. coli with DNase activities. The sequence of the 1295-bp polynucleotide from a clone of the bpcDNA was deduced by DNA sequencing.…”

Section: The Primary Secondary and Tertiary Structuresmentioning

confidence: 99%

Structure and Function of Bovine Pancreatic Deoxyribonuclease I

Chen¹,

Liao

2006

PPL

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Bovine pancreatic deoxyribonuclease I (bpDNase), the first DNase discovered, is the best characterized among various types of DNase. A catalytic mechanism has been suggested based on the X-ray structure of the bpDNase-octamer complex. In this review, we will focus on three aspects: 1) the distinctive functions of the two structural calcium atoms; 2) the biological functions of the two disulfides; and 3) the involvement of the N- and C-terminal fragments in the enzyme folding for activity.

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“…This cDNA was composed of a 48 bp 5′‐UTR, an 855 bp ORF and a 95 bp 3′‐UTR. Thus, shark DNase I cDNAs appear to be characterized by a shorter 3′‐UTR (average of 96 bp) than those cloned from most other vertebrates (200 ± 89 bp), including the human [29], rabbit [13], mouse [14], rat [30], cow [31], hen [17], pig [15] and snake [18] enzymes. In this respect, shark DNases I resemble the amphibian (89 ± 22 bp) [19] and Osteichtye (112 ± 20 bp) [20,21] enzymes.…”

Section: Cdna Constructs Encoding Shark Dnases Imentioning

confidence: 99%

Molecular evolution of shark and other vertebrate DNases I

Yasuda¹,

Iida

Ueki³

et al. 2004

European Journal of Biochemistry

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We purified pancreatic deoxyribonuclease I (DNase I) from the shark Heterodontus japonicus using three‐step column chromatography. Although its enzymatic properties resembled those of other vertebrate DNases I, shark DNase I was unique in being a basic protein. Full‐length cDNAs encoding the DNases I of two shark species, H. japonicus and Triakis scyllia, were constructed from their total pancreatic RNAs using RACE. Nucleotide sequence analyses revealed two structural alterations unique to shark enzymes: substitution of two Cys residues at positions 101 and 104 (which are well conserved in all other vertebrate DNases I) and insertion of an additional Thr or Asn residue into an essential Ca2+‐binding site. Site‐directed mutagenesis of shark DNase I indicated that both of these alterations reduced the stability of the enzyme. When the signal sequence region of human DNase I (which has a high α‐helical structure content) was replaced with its amphibian, fish and shark counterparts (which have low α‐helical structure contents), the activity expressed by the chimeric mutant constructs in transfected mammalian cells was approximately half that of the wild‐type enzyme. In contrast, substitution of the human signal sequence region into the amphibian, fish and shark enzymes produced higher activity compared with the wild‐types. The vertebrate DNase I family may have acquired high stability and effective expression of the enzyme protein through structural alterations in both the mature protein and its signal sequence regions during molecular evolution.

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Cloning, sequencing and expression of a cDNA encoding bovine pancreatic deoxyribonuclease I in Escherichia coli: purification and characterization of the recombinant enzyme

Cited by 23 publications

References 13 publications

The distinctive functions of the two structural calcium atoms in bovine pancreatic deoxyribonuclease

The distinctive functions of the two structural calcium atoms in bovine pancreatic deoxyribonuclease

Structure and Function of Bovine Pancreatic Deoxyribonuclease I

Molecular evolution of shark and other vertebrate DNases I

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