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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

Staining of Glycoproteins in SDS-Polyacrylamide Gels.(A). SDS-PAGE of RKOD with or without digestion with Endo H. M, protein molecular weight marker (the size of each band is indicated on the left); lane 1, RKOD; lane 2, RKOD treated with Endo H; lane 3, denatured RKOD treated with Endo H; lane 4, denatured RKOD; lane 5, positive control (Horseradish Peroxidase) from the kit; and lane 6, negative control (Soybean Trypsin Inhibitor) from the kit. (B). Glycoprotein staining of RKOD with or without Endo H digestion. All samples were loaded in the same order as A.
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pone.0131757.g003: Staining of Glycoproteins in SDS-Polyacrylamide Gels.(A). SDS-PAGE of RKOD with or without digestion with Endo H. M, protein molecular weight marker (the size of each band is indicated on the left); lane 1, RKOD; lane 2, RKOD treated with Endo H; lane 3, denatured RKOD treated with Endo H; lane 4, denatured RKOD; lane 5, positive control (Horseradish Peroxidase) from the kit; and lane 6, negative control (Soybean Trypsin Inhibitor) from the kit. (B). Glycoprotein staining of RKOD with or without Endo H digestion. All samples were loaded in the same order as A.

Mentions: According to the online Prediction Servers NetNGlyc 1.0 and NetOGlyc 4.0 (http://www.cbs.dtu.dk/services/NetNGlyc/ and http://www.cbs.dtu.dk/services/NetOGlyc/), RKOD has one putative N-linked glycosylation site (NSV) and six putative O-linked glycosylation sites. Thus, the variation in the apparent molecular mass of RKOD observed by SDS-PAGE may be attributable to glycosylation. Endo H is commonly used to identify the composition of the glycan portion of N-glycosylated proteins [19–20]. To test our hypothesis, RKOD was treated with Endo H under both native and denaturing condition, followed by staining with the GelCode Glycoprotein Staining Kit to detect glycoprotein sugar moieties. As clearly demonstrated in Fig 3, the upper band of RKOD appeared as a magenta band, indicating glycosylation of RKOD.


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)

Staining of Glycoproteins in SDS-Polyacrylamide Gels.(A). SDS-PAGE of RKOD with or without digestion with Endo H. M, protein molecular weight marker (the size of each band is indicated on the left); lane 1, RKOD; lane 2, RKOD treated with Endo H; lane 3, denatured RKOD treated with Endo H; lane 4, denatured RKOD; lane 5, positive control (Horseradish Peroxidase) from the kit; and lane 6, negative control (Soybean Trypsin Inhibitor) from the kit. (B). Glycoprotein staining of RKOD with or without Endo H digestion. All samples were loaded in the same order as A.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131757.g003: Staining of Glycoproteins in SDS-Polyacrylamide Gels.(A). SDS-PAGE of RKOD with or without digestion with Endo H. M, protein molecular weight marker (the size of each band is indicated on the left); lane 1, RKOD; lane 2, RKOD treated with Endo H; lane 3, denatured RKOD treated with Endo H; lane 4, denatured RKOD; lane 5, positive control (Horseradish Peroxidase) from the kit; and lane 6, negative control (Soybean Trypsin Inhibitor) from the kit. (B). Glycoprotein staining of RKOD with or without Endo H digestion. All samples were loaded in the same order as A.
Mentions: According to the online Prediction Servers NetNGlyc 1.0 and NetOGlyc 4.0 (http://www.cbs.dtu.dk/services/NetNGlyc/ and http://www.cbs.dtu.dk/services/NetOGlyc/), RKOD has one putative N-linked glycosylation site (NSV) and six putative O-linked glycosylation sites. Thus, the variation in the apparent molecular mass of RKOD observed by SDS-PAGE may be attributable to glycosylation. Endo H is commonly used to identify the composition of the glycan portion of N-glycosylated proteins [19–20]. To test our hypothesis, RKOD was treated with Endo H under both native and denaturing condition, followed by staining with the GelCode Glycoprotein Staining Kit to detect glycoprotein sugar moieties. As clearly demonstrated in Fig 3, the upper band of RKOD appeared as a magenta band, indicating glycosylation of RKOD.

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