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 | 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 | 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 | UniProt | Comment | Textmining |
---|---|---|---|
Thermococcus kodakarensis KOD1 | P77933 | - |
- |
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 (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 | Comment | Organism |
---|---|---|
RKOD DNA polymerase | - |
Thermococcus kodakarensis KOD1 |
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 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 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 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 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 |