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Expression and Characterization of the RKOD DNA Polymerase in Pichia pastoris.

Wang F, Li S, Zhao H, Bian L, Chen L, Zhang Z, Zhong X, Ma L, Yu X - PLoS ONE (2015)

Bottom Line: The resulting novel gene was cloned into a pHBM905A vector and introduced into P. pastoris GS115 for secretory expression.The recombinant KOD expressed in P. pastoris exhibited excellent thermostability, extension rate and fidelity.This is the first report of the expression in yeast of a DNA polymerase for use in PCR.

View Article: PubMed Central - PubMed

Affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan, People's Republic of China.

ABSTRACT
The present study assessed high-level expression of the KOD DNA polymerase in Pichia pastoris. Thermococcus kodakaraensis KOD1 is a DNA polymerase that is widely used in PCR. The DNA coding sequence of KOD was optimized based on the codon usage bias of P. pastoris and synthesized by overlapping PCR, and the nonspecific DNA-binding protein Sso7d from the crenarchaeon Sulfolobus solfataricus was fused to the C-terminus of KOD. The resulting novel gene was cloned into a pHBM905A vector and introduced into P. pastoris GS115 for secretory expression. The yield of the target protein reached approximately 250 mg/l after a 6-d 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 was purified, and its enzymatic features were studied. Its specific activity was 19,384 U/mg. The recombinant KOD expressed in P. pastoris exhibited excellent thermostability, extension rate and fidelity. Thus, this report provides a simple, efficient and economic approach to realize the production of a high-performance thermostable DNA polymerase on a large scale. This is the first report of the expression in yeast of a DNA polymerase for use in PCR.

No MeSH data available.


Related in: MedlinePlus

Effects of temperature, pH and Mg2+ on RKOD DNA polymerase activity.(A) Effect of pH on DNA polymerase activity. (B) Effect of Mg2+ on DNA polymerase activity. (C) Effect of temperature on DNA polymerase activity. The standard assay described in the text was performed with 1 U of purified DNA polymerase. These experiments were repeated for 3 times.
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pone.0131757.g002: Effects of temperature, pH and Mg2+ on RKOD DNA polymerase activity.(A) Effect of pH on DNA polymerase activity. (B) Effect of Mg2+ on DNA polymerase activity. (C) Effect of temperature on DNA polymerase activity. The standard assay described in the text was performed with 1 U of purified DNA polymerase. These experiments were repeated for 3 times.

Mentions: Next, we determined the characteristics of the RKOD DNA polymerase (i.e., optimum pH, temperature, and required concentration of metal ions). RKOD exhibited maximum activity at pH 8.5 (Fig 2A). Similar to all other DNA polymerases, RKOD required a divalent cation as a cofactor; optimal activity was obtained with 2.0 mM MgSO4 (Fig 2B). The optimal temperature for the catalytic activity of RKOD was (Fig 2C) 70–75°C.


Expression and Characterization of the RKOD DNA Polymerase in Pichia pastoris.

Wang F, Li S, Zhao H, Bian L, Chen L, Zhang Z, Zhong X, Ma L, Yu X - PLoS ONE (2015)

Effects of temperature, pH and Mg2+ on RKOD DNA polymerase activity.(A) Effect of pH on DNA polymerase activity. (B) Effect of Mg2+ on DNA polymerase activity. (C) Effect of temperature on DNA polymerase activity. The standard assay described in the text was performed with 1 U of purified DNA polymerase. These experiments were repeated for 3 times.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4489709&req=5

pone.0131757.g002: Effects of temperature, pH and Mg2+ on RKOD DNA polymerase activity.(A) Effect of pH on DNA polymerase activity. (B) Effect of Mg2+ on DNA polymerase activity. (C) Effect of temperature on DNA polymerase activity. The standard assay described in the text was performed with 1 U of purified DNA polymerase. These experiments were repeated for 3 times.
Mentions: Next, we determined the characteristics of the RKOD DNA polymerase (i.e., optimum pH, temperature, and required concentration of metal ions). RKOD exhibited maximum activity at pH 8.5 (Fig 2A). Similar to all other DNA polymerases, RKOD required a divalent cation as a cofactor; optimal activity was obtained with 2.0 mM MgSO4 (Fig 2B). The optimal temperature for the catalytic activity of RKOD was (Fig 2C) 70–75°C.

Bottom Line: The resulting novel gene was cloned into a pHBM905A vector and introduced into P. pastoris GS115 for secretory expression.The recombinant KOD expressed in P. pastoris exhibited excellent thermostability, extension rate and fidelity.This is the first report of the expression in yeast of a DNA polymerase for use in PCR.

View Article: PubMed Central - PubMed

Affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan, People's Republic of China.

ABSTRACT
The present study assessed high-level expression of the KOD DNA polymerase in Pichia pastoris. Thermococcus kodakaraensis KOD1 is a DNA polymerase that is widely used in PCR. The DNA coding sequence of KOD was optimized based on the codon usage bias of P. pastoris and synthesized by overlapping PCR, and the nonspecific DNA-binding protein Sso7d from the crenarchaeon Sulfolobus solfataricus was fused to the C-terminus of KOD. The resulting novel gene was cloned into a pHBM905A vector and introduced into P. pastoris GS115 for secretory expression. The yield of the target protein reached approximately 250 mg/l after a 6-d 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 was purified, and its enzymatic features were studied. Its specific activity was 19,384 U/mg. The recombinant KOD expressed in P. pastoris exhibited excellent thermostability, extension rate and fidelity. Thus, this report provides a simple, efficient and economic approach to realize the production of a high-performance thermostable DNA polymerase on a large scale. This is the first report of the expression in yeast of a DNA polymerase for use in PCR.

No MeSH data available.


Related in: MedlinePlus