Any feedback?
Please rate this page
(literature.php)
(0/150)

BRENDA support

Literature summary for 2.7.7.7 extracted from

  • Wang, F.; Li, S.; Zhao, H.; Bian, L.; Chen, L.; Zhang, Z.; Zhong, X.; Ma, L.; Yu, X.
    Expression and characterization of the RKOD DNA polymerase in Pichia pastoris (2015), PLoS One, 10, e0131757.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
high-level expression in Pichia pastoris. The DNA coding sequence of KOD from Pyrococcus sp. KOD1 (Gene Bank: D29671) is optimized based on the codon usage bias of Pichia pastoris and synthesized by overlapping PCR, and the nonspecific DNA-binding protein Sso7d from Sulfolobus solfataricus is fused to the C-terminus of KOD. The resulting novel gene is cloned into a pHBM905A vector and introduced into Pichia pastoris GS115 for secretory expression. The yield of the target protein reaches approximately 250 mg/l after a 6 day induction with 1% (v/v) methanol in shake flasks. This yield is much higher than those of other DNA polymerases expressed heterologously in Escherichia coli. The recombinant enzyme expressed in Pichia pastoris exhibits excellent thermostability, extension rate and fidelity Thermococcus kodakarensis KOD1

Protein Variants

Protein Variants Comment Organism
additional information the DNA coding sequence of KOD from Pyrococcus sp. KOD1 is optimized based on the codon usage bias of Pichia pastoris and synthesized by overlapping PCR, and the nonspecific DNA-binding protein Sso7d from Sulfolobus solfataricus is fused to the C-terminus of KOD. The resulting novel gene is cloned into a pHBM905A vector and introduced into Pichia pastoris GS115 for secretory expression. The yield of the target protein reaches approximately 250 mg/l after a 6 day induction with 1% (v/v) methanol in shake flasks. This yield is much higher than those of other DNA polymerases expressed heterologously in Escherichia coli. The recombinant enzyme expressed in Pichia pastoris exhibits excellent thermostability, extension rate and fidelity. When Sso7d is fused to the enzyme, a significant enhancement of processivity is achieved regardless of the starting processivity of the enzyme Thermococcus kodakarensis KOD1

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ requires a divalent cation, optimal activity is obtained with 2.0 mM MgSO4, recombinant enzyme with the nonspecific DNA-binding protein Sso7d from Sulfolobus solfataricus fused to the C-terminus Thermococcus kodakarensis KOD1

Organism

Organism UniProt Comment Textmining
Thermococcus kodakarensis KOD1 P77933
-
-

Posttranslational Modification

Posttranslational Modification Comment Organism
glycoprotein the recombinant enzyme with the nonspecific DNA-binding protein Sso7d from Sulfolobus solfataricus fused to the C-terminus is a glycoprotein Thermococcus kodakarensis KOD1

Purification (Commentary)

Purification (Comment) Organism
the DNA coding sequence of KOD from Pyrococcus sp. KOD1 (Gene Bank: D29671) is optimized based on the codon usage bias of Pichia pastoris and synthesized by overlapping PCR, and the nonspecific DNA-binding protein Sso7d from Sulfolobus solfataricus is fused to the C-terminus of KOD. The resulting novel gene is cloned into a pHBM905A vector and introduced into Pichia pastoris GS115 for secretory expression. The yield of the target protein reaches approximately 250 mg/l after a 6 day induction with 1% (v/v) methanol in shake flasks. This yield is much higher than those of other DNA polymerases expressed heterologously in Escherichia coli. The recombinant enzyme is purified Thermococcus kodakarensis KOD1

Synonyms

Synonyms Comment Organism
RKOD DNA polymerase
-
Thermococcus kodakarensis KOD1

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
70 75 recombinant enzyme with the nonspecific DNA-binding protein Sso7d from Sulfolobus solfataricus fused to the C-terminus Thermococcus kodakarensis KOD1

Temperature Range [°C]

Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
60 80 60°C: about 45% of maximal activity, 80°C: about 50% of maximal activity, recombinant enzyme with the nonspecific DNA-binding protein Sso7d from Sulfolobus solfataricus fused to the C-terminus Thermococcus kodakarensis KOD1

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
95
-
21 h, the enzyme retains polymerase activity Thermococcus kodakarensis KOD1
100
-
11 h, recombinant enzyme with the DNA-binding protein Sso7d from Sulfolobus solfataricus fused to the C-terminus retains activity Thermococcus kodakarensis KOD1

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8.5
-
recombinant enzyme with the nonspecific DNA-binding protein Sso7d from Sulfolobus solfataricus fused to the C-terminus Thermococcus kodakarensis KOD1

pH Range

pH Minimum pH Maximum Comment Organism
6.5 9 pH 6.5: about 55% of maximal activity, pH 9.0: about 90% of maximal activity, recombinant enzyme with the nonspecific DNA-binding protein Sso7d from Sulfolobus solfataricus fused to the C-terminus Thermococcus kodakarensis KOD1