Expression and Characterization of the RKOD DNA Polymerase in Pichia pastoris

Fei Wang; Shuntang Li; Hui Zhao; Lu Bian; Liang Chen; Zhen Zhang; Xing Zhong; Lixin Ma; Xiaolan Yu

doi:10.1371/journal.pone.0131757

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.

Citation: Wang F, Li S, Zhao H, Bian L, Chen L, Zhang Z, et al. (2015) Expression and Characterization of the RKOD DNA Polymerase in Pichia pastoris. PLoS ONE 10(7): e0131757. https://doi.org/10.1371/journal.pone.0131757

Editor: Eugene A. Permyakov, Russian Academy of Sciences, Institute for Biological Instrumentation, RUSSIAN FEDERATION

Received: January 29, 2015; Accepted: June 5, 2015; Published: July 2, 2015

Copyright: © 2015 Wang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: This work was supported by grants No. 2013CB910801: http://english.iee.cas.cn/rh/rps/201005/t20100510_53822.html, No. 2012FFA034, http://www.hbstd.gov.cn/index.htm, No.2014070504020239, http://www.whst.gov.cn/, No.31172320 and 31340036, http://www.physics.sjtu.edu.cn/en/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

More than 50 DNA polymerases have been cloned and sequenced from various organisms. Most native thermostable enzymes are synthesized at low levels by thermophilic bacteria and consequently are difficult to purify. Several thermostable DNA polymerases have been produced in a biologically active form using Escherichia coli and baculovirus systems. The Tay, Pfu, Pwo and Taq DNA polymerases were expressed in E. coli at expression levels of approximately 2.25 mg/l, 24 mg/l, 26.6 mg/l, and 95 mg/l, respectively [1–3]. However, the lower soluble yields were not satisfactory. The Pfu DNA polymerase was also expressed as a secreted protein in Spodoptera frugiperda cells (sf-9) and Trichoplusia ni cells (hi-5) at expression levels of approximately 100 mg/l and 134 mg/l, respectively [4]. Baculovirus expression enables the production of commercial quantities but is more difficult and expensive than bacterial expression.

Increasing the output and simplifying the purification steps of thermostable DNA polymerases have recently been emphasized [5]. Pichia pastoris is an established protein expression host that is mainly applied for the production of biopharmaceuticals and industrial enzymes. This methylotrophic yeast can grow to very high cell densities and thus represents a unique production system for strong and tightly regulated promoters that can produce gram amounts of recombinant proteins per liter of culture both intracellularly and in a secretory fashion [6].

The KOD DNA polymerase from the archaeon Pyrococcus sp. strain KOD1 exhibits 5-fold higher extension rate (100 to 130 nucleotides/s) and 10- to 15-fold higher processivity (persistence of sequential nucleotide polymerization) compared to the DNA polymerase from Pyrococcus furiosus (Pfu DNA polymerase), and the mutation frequency (3.5 ×10⁻³) was similar to Pfu DNA polymerase. Due to these characteristics, KOD DNA polymerase can be used to perform more accurate PCR with a shorter reaction time. In the present study, we report the high-level secretory expression of KOD DNA polymerase in P. pastoris for the first time.

The dsDNA binding protein Sso7d from Sulfolobus solfataricus has been reported to significantly enhance the processivity of KOD polymerase [7]. Therefore, in this study, the Sso7d gene was fused to the KOD C-terminus to enhance the processivity.

Materials and Methods

Strains, vectors, media and reagents

P. pastoris GS115 was obtained from Invitrogen (USA). E. coli XL10-Gold was purchased from Stratagene (USA). The pMD18-T vector was purchased from Takara (JPN). The yeast expression vector pHBM905A (ColE1 ori, Amp^r, Kan^r, HIS4, P_AOX1, T_AOX1) was synthesized previously in our laboratory [8]. The vector pBAD-His-GFP was constructed previously [9]. Luria-Bertani (LB) medium was prepared for the cultivation of E. coli as described in the Manual of Molecular Cloning [10]. For yeast culture, buffered glycerol-complex medium (BMGY), buffered methanol-complex medium (BMMY) and minimal dextrose medium (MD) were prepared as specified in the Pichia Expression Kit (Invitrogen, USA). Endoglycosidase H (Endo H) was purchased from NEB (USA). The DNA Gel Extraction Kit was from V-gene (USA). Restriction endonucleases (CopI, NotI, and SalI), DpnI, T4 DNA polymerase, and the Prime Star DNA polymerase were purchased from TaKaRa (JPN). KOD plus was purchased from Toyobo (JPN). dATP, dCTP, dGTP, and dTTP were from Thermo Scientific (USA). [α-³²P]-dCTP was from PerkinElmer (USA). Primers were synthesized by Sangon Biotech (China). All other reagents used in this study were of analytical grade.

Design and synthesis of the KOD and Sso7d genes

The DNA coding sequences of KOD from Pyrococcus sp. KOD1 (Gene Bank: D29671) and Sso7d from S. solfataricus P2 (Gene ID: 1454006) were optimized to match the codon usage preference of Pichia pastoris. Sso7d was fused to the C-terminus of KOD. To synthesize this modified ORF, 72 oligonucleotides (S1 Table) were designed for overlapping PCR using the DNAWorks program (http://helixweb.nih.gov/dnaworks). The gene synthesis procedure was performed as described previously [11]. The novel DNA fragment was cloned into pMD18-T and confirmed by DNA sequencing (Sangon, China).

Constructing the expression vector for KOD expression in Pichia pastoris GS115 and E. coli

For expression in Pichia pastoris, the target gene was amplified from the recombinant T-vector using the primers RKOD-1 and RKOD-72 (S1 Table). The resulting PCR product was treated with T4 DNA polymerase at 12°C for 20 min in the presence of 1 mM dTTP and ligated into pHBM905A vector digested with NotI and CpoI [12].

For expression in E. coli, the primers Kod-F and Kod-R and Kod-F and Kod-S-R were used to amplify KOD and KOD-Sso from the recombinant T-vectors. The resulting PCR products were ligated into pBAD-His-GFP vector digested with Bfu I [9].

Expression and purification of RKOD in Pichia pastoris

For expression in P. pastoris, the plasmid pHBM-RKOD was linearized using SalI and used to transform P. pastoris GS115 via electroporation (4 kΩ, 50μF, and 400 V). Transformants were screened on MD plates containing 0.4 mg/l biotin without histidine and identified by PCR using the primer pair RKOD-1 and RKOD-72. Recombinant Pichia pastoris GS115 bearing the target gene in its genome was incubated in 100 ml BMGY until the OD₆₀₀ reached approximately 20. All cells were harvested by centrifugation and transferred to 25 ml of BMMY. A total of 1% (v/v) methanol was added every 24 h to induce target protein expression. Approximately 1 ml of cell culture was collected every 24 h and centrifuged at 6,000×g for 5 min to remove cells. After 168 h, an equal volume of each supernatant (15 μl) was loaded onto a 12% (w/v) polyacrylamide gel for SDS-PAGE according to the procedure of Laemmli [13], followed by staining with Coomassie Brilliant Blue G-250. The total protein concentration in each supernatant was measured using the Micro-BCA Protein Assay Reagent (Pierce, USA).

After induction with methanol for 120 h, the target protein was purified by centrifuging approximately 20 ml of BMMY cell culture at 6,000×g for 5 min. Then, the supernatant was immersed in a 75°C water orbital shaker and shaken for 30 min, cooled on ice for 20 min, and then centrifuged at 10,000×g at 4°C for 20 min. The clarified supernatant was collected and dialyzed in a Millipore 30-kDa cut-off membrane at 4°C to remove ions and salts. The sample was resuspended in 15 ml of Buffer A (20 mM Tris-HCl, 0.1 M NaCl, and 0.1 mM EDTA, pH 7.5) and centrifuged at 5000×g to achieve a final volume of 1 ml. This step was repeated twice.

Expression and purification of KOD and KOD-Sso in E. coli

Plasmid encoding KOD and KOD-Sso was used to transform E. coli strain BL21 (Stratagene), which was grown in 500 ml of LB medium in the presence of carbenicillin to an OD600 of 0.5. Arabinose was added at a concentration of 1 mg/ml to induce expression from the ara promoter.

Cells were harvested 7 h later by centrifugation, and the pellet was resuspended in 20 ml of binding buffer (20 mM Tris-HCl, pH 7.9, 0.5 M NaCl, and 5 mM imidazole). The cells were disrupted by sonication, and the insoluble debris was removed by centrifugation. For purification, the cleared lysate was immersed in a 75°C water orbital shaker and shaken for 30 min, cooled on ice for 20 min, and then centrifuged at 10,000×g at 4°C for 20 min to remove the denatured E. coli proteins. A His-bind resin and His-bind buffer kit (Novagen, Madison, WI, USA) were used to purify the His-tagged protein according to Novagen’s instructions. The protein was eluted from the resin in 4 ml of elution buffer (20 mM Tris-HCl, pH 7.9, 100 mM imidazole, and 0.5 M NaCl). The protein concentration was measured using the Micro-BCA Protein Assay Reagent (Pierce, USA). The active fractions were pooled and dialyzed against storage buffer (20 mM Tris-HCl, 0.1 M NaCl, and 0.1 mM EDTA, pH 7.5) at 4°C overnight.

DNA polymerase assays

DNA polymerase activity was assayed essentially as described by Grippo and Richardson [14]. The 50-μl reaction mixture contained 5 μl of 10×RKOD buffer (100 mM Tris-HCl, pH 8.8, 10 mM (NH₄)₂SO₄, 100 mM KCl, 20 mM MgSO₄, 0.1% Triton-100, and 1 mg/ml bovine serum albumin), 0.5 mM heat-denatured salmon sperm DNA, 0.20 mM each dCTP, dATP, dTTP, and dGTP, and 1 μCi of [α-32P]dCTP. The assay reactions were incubated at 72°C for 30 min and terminated on ice; 5-μl aliquots were then spotted onto ion-exchange paper discs (DE-81, Whatman). The spotted discs were dried, washed three times with 2x SSC buffer (300 mM sodium chloride and 30 mM trisodium citrate, pH 7.0) for 5 min each, washed once in 100% cold ethanol, and air-dried. The incorporated radioactivity was counted using a liquid scintillation counter. KOD-Plus DNA polymerase (0.5–2.5 units) was used as a positive control. One unit of RKOD DNA polymerase is defined as the amount of RKOD polymerase that incorporates 10 nmol dNTPs into a DE81-bound form at 72°C in 30 min.

The optimal reaction conditions for the recombinant RKOD DNA polymerase were used in the DNA polymerase assay as described previously. RKOD activity was measured under different pH, MgSO₄ concentration, and temperature conditions.

Staining Glycoproteins in SDS-Polyacrylamide Gels

Glycoprotein staining was performed using the Thermo Scientific Pierce Glycoprotein Staining Kit in accordance with the manufacturer’s recommendations. An equal amount of each sample was loaded onto two 12% (w/v) polyacrylamide gels. After SDS-PAGE, one gel was stained with Coomassie Brilliant Blue G-250, and the other was stained using the Glycoprotein Staining Kit.

Thermostability analysis

Temperature stability was assayed as described by Dabrowski S, and J Kur [2]. Purified RKOD, KOD and KOD-Sso were heated at 95°C and 100°C from 1 h to 21 h. The same amount of enzyme was used to amplify an approximately 1-kb target fragment under the same reaction conditions. Each PCR reaction was performed in a 25-μl mixture containing 2.5 μl of 10× RKOD buffer (100 mM Tris-HCl, pH 8.8, 10 mM (NH₄)₂SO₄, 100 mM KCl, 20 mM MgSO_4, 0.1% Triton-100, and 1 mg/ml bovine serum albumin), 10 pmol of primer P905-F and P1000-R (S1 Table), 0.01 μg of the pHBM905A plasmid, and 0.2 mM each dATP, dGTP, dCTP and dTTP. The PCR cycling parameters were as follows: 2 min of denaturation at 95°C, 25 cycles of 10 s at 95°C, 15 s at 56°C, and 30 s at 72°C, ending with an additional 7-min elongation at 72°C. Equal volumes of PCR products (4 μl) were analyzed by agarose gel electrophoresis.

Fidelity of DNA polymerase

A PCR-based forward mutation assay was performed as described by Kelly S. Lundberg et al [15], except that “mannanase activity” was employed to measure the error rate of the RKOD DNA polymerase. The mannanase gene was amplified from pYEV–man [16] using the primers man-F and man-R (S1 Table) and cloned into the BfuI site of pBAD-His-GFP; the resulting construct is referred to as pBAD-mannanase. The plasmid pBAD-mannanase was amplified by inverse PCR [17] with the primer pair p-man-F and p-man-R (S1 Table) and contained a homologous sequence of 18 base pairs. PCR was performed with the RKOD, KOD plus, and Prime Star DNA polymerases under optimized conditions for each polymerase. The resulting PCR products were treated with DpnI, subjected to agarose gel DNA purification, and transformed into E. coli XL10-Gold. These clones were transferred to LB plates containing 100 mM arabinose and 0.03% (m/v) trypan blue (which gives the plates a blue appearance) and 0.5% (m/v) Konjac Flour as the substrate for the mannanase. A clear zone was visible around mannanase-expressing colonies capable of hydrolyzing the Konjac glucomannan [16–18]. A single colony of E. coli XL10-Gold that contained the plasmid pBAD-man was used as a positive control, and a single colony of E. coli XL10-Gold that contained the plasmid pBAD-His-GFP was used as a negative control. Hydrolysis holes with different sizes were observed and measured after 12 hours of culture. Error rates were calculated based on the sizes of the hydrolysis holes. Clones that produced much larger or smaller hydrolysis holes were considered mutants.

PCR efficiency assay

In this experiment, plasmid pHBM905A targets (2–9 kb) were amplified using a range of extension times to compare the efficiencies of RKOD, KOD and KOD-S. The plasmid pHBM905A was used as the template. The primer pairs P905-F and P2000-R (for 2 kb), P905-F and P4000-R (for 4 kb), P905-F and P6000-R (for 6 kb) and P905-F and P9000-R (for 9 kb) (S1 Table) were designed to amplify targets with lengths of 2 kb, 4 kb, 6 kb, and 9 kb, respectively. Each PCR reaction was performed in a 50-μl mixture containing 5 μl of 10× RKOD buffer (100 mM Tris-HCl, pH 8.8, 10 mM (NH₄)₂SO₄, 100 mM KCl, 20 mM MgSO₄, 0.1% Triton-100, and 1 mg/ml bovine serum albumin), 10 pmol each primer, 0.01 μg of the pHBM905A plasmid, 0.2 mM each dATP, dGTP, dCTP and dTTP, and purified RKOD, KOD and KOD-Sso. The PCR cycling parameters were as follows: 2 min denaturation at 95°C, 25 cycles of 10 s at 95°C, 15 s at 56°C, and 30–50 sec at 72°C, ending with an additional 7-min elongation at 72°C. Equal volumes of PCR products (4 μl) were analyzed using 1% agarose gel electrophoresis.

Results

Optimization and synthesis of the RKOD gene

The KOD gene of Pyrococcus sp. strain KOD1 contains a 5013-bp open reading frame (GenBank: D29671); the entire mature KOD DNA polymerase encodes a 90-kDa protein. The Sso7d gene from S. solfataricus P2 (Gene ID: 1454006) is 207 bp in length and encodes a 7-kD protein. These sequences were modified based on the codon usage bias of P. pastoris without changing the amino acid sequence and synthesized by overlapping PCR. The Sso7D gene was fused to the C-terminus of KOD. For expression in P. pastoris, the KOD-Sso fragment was cloned into the pHBM905A vector fused with the MF-α leader sequence. The recombinant plasmid was confirmed by DNA sequencing and designated pHBM-RKOD. For expression in E. coli, KOD and KOD-Sso were cloned into the pBAD-His-GFP vector fused with the 6xHis tag under the control of the ara promoter. The recombinant plasmids were confirmed by DNA sequencing and termed pBAD-KOD and pBAD-KOD-S, respectively.

Expression and purification of the recombinant KOD DNA polymerase

Positive colonies were selected randomly to assess the expression of RKOD. SDS-PAGE of the supernatants revealed the presence of three bands from the first day of induction. (Fig 1). All bands were identified as the target protein RKOD by mass spectrometry (MS) (data not shown). The total amount of protein in the supernatant was 150, 181, 192 and 230 mg/l on days 1, 2, 3, 4, respectively. The expression of RKOD reached a maximum level at 120 h, with a protein concentration of approximately 250 mg/l. RKOD expression decreased thereafter to 239 mg/l at 144 h. These results demonstrated the successful expression of RKOD in P. pastoris. The recombinant protein was designated RKOD. The fermentation supernatant was purified, and the protein concentration was 180 mg/l after purification.

Soluble KOD and KOD-S expressed in E. coli were purified by Ni-chelate affinity chromatography. The protein concentrations of KOD and KOD-S were 25 mg/l and 20 mg/l after purification, respectively.

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Fig 1. SDS-PAGE analysis of secreted RKOD in the cell culture supernatants of the shake flasks and soluble KOD and KOD-S expressed in E. coli.

(A). M, protein molecular weight marker (the size of each band is indicated on the left); lane 1, cell culture supernatant (15 μl) of the strain bearing the pHBM905A plasmid (negative control); lanes 2–7, cell culture supernatants (15 μl) collected from days 1–6, respectively; and lane 8, purified RKOD; (B). M, protein molecular weight marker (the size of each band is indicated on the left); lane 1, purified KOD; lane 2, purified KOD-S.

https://doi.org/10.1371/journal.pone.0131757.g001

Activity assessment of recombinant RKOD

The enzymatic activities of purified RKOD, KOD and KOD-S were approximately 19,384 U/mg, 20,500 U/mg and 20,100 U/mg, respectively. These results indicate that Sso7d fusion did not negatively affect the catalytic activity of the KOD DNA polymerase.

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.

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Fig 2. 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.

https://doi.org/10.1371/journal.pone.0131757.g002

Glycosylation analysis of RKOD

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.

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Fig 3. 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.

https://doi.org/10.1371/journal.pone.0131757.g003

Temperature stability assay

Protein glycosylation is one of the most common covalent post-translational modifications and has a significant effect on the structure and function of proteins, particularly thermostability [21–22]. A PCR-based assay was used to analyze the thermostability of KOD, KOD-S and RKOD. Gel imaging analysis revealed that KOD, KOD-S and RKOD retained their polymerase activity after incubation at 95°C for 21 h (Fig 4A). Following incubation at 100°C for 11 h, (Fig 4B), RKOD retained polymerase activity, but the activities of KOD and KOD-S were reduced. These results indicate RKOD is more thermostable than KOD and KOD-S, possibly due to the glycosylation of RKOD. However, the thermal stability profile of the KOD-S fusion protein was nearly identical to that of KOD. These results demonstrate that the fusion of Sso7d to the polymerase does not negatively affect the thermal stability of the polymerase domain.

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Fig 4. Thermostability of RKOD expressed in Pichia pastoris.

Residual activity was assayed by PCR after incubation at 95°C or 100°C. (A). Thermostability of the KOD, KOD-S and RKOD DNA polymerase at 95°C. M, DL2000 marker (the size of each band is indicated on the left); lane 1, PCR with untreated KOD, KOD-S and RKOD (positive control); lanes 2–12, PCR with KOD, KOD-S and RKOD incubated at 95°C for 1–21 h. (B). Thermostability of the KOD, KOD-S and RKOD DNA polymerase at 100°C. Lane 1, PCR with untreated KOD, KOD-S and RKOD (positive control); lanes 2–9, PCR with KOD, KOD-S and RKOD incubated at 100°C for 1–15 h.

https://doi.org/10.1371/journal.pone.0131757.g004

PCR error rate of the RKOD DNA polymerase

High-fidelity PCR enzymes are valuable for minimizing the introduction of amplification errors in products that will be cloned, sequenced, and expressed [23]. Therefore, maintaining the high fidelity of the RKOD polymerase obtained from different expression systems is important. RKOD expressed in P. pastoris had an error rate of 0.38%, similar to the error rates of commercial high-fidelity PCR enzymes (Table 1).

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Table 1. Comparison of the fidelities of thermostable DNA polymerases.

https://doi.org/10.1371/journal.pone.0131757.t001

PCR efficiency assay

The major uses of thermostable DNA polymerases are for in vitro amplification of DNA fragments and DNA sequencing. The ultimate goal of improving the processivity of thermostable DNA polymerases is to improve their performance in vitro, such as in PCR or cycle sequencing applications [7]. Because an increase in processivity allows the polymerase to incorporate more nucleotides per binding event, it should also enable more efficient in vitro replication of the template strand during each PCR cycle. Consequently, shorter extension times may be required to amplify the same target or a much longer target could be amplified with the same extension time. In this experiment, the plasmid pHBM905A (2–9 kb) was amplified using a range of extension times to compare the efficiencies of KOD, KOD-S and RKOD (Fig 5). A 50-sec/cycle extension time was required to amplify a 9-kb target when 60 U/ml KOD was used. By contrast, when 20U/ml KOD-S and RKOD were used the same 9-kb target could be amplified with a 30 s/cycle extension time. These findings demonstrate that when Sso7d is fused to KOD, a significant enhancement of processivity is achieved regardless of the starting processivity of the enzyme. When KOD-S and RKOD are tested in PCR amplifications, a clear advantage over KOD is observed. Not only is much less enzyme required, but a much shorter extension time can be used.

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Fig 5. Comparison of the PCR efficiencies of KOD, KOD-S and RKOD.

The plasmid pHBM905A was used as the template, and the sizes of the amplicons are indicated at the bottom. M, λ/EcoR T14 marker; lanes 1–4, the target lengths of 2 kb, 4 kb, 6 kb, and 9 kb are marked. PCR buffer 10 mM Tris-HCl, pH 8.8, 1 mM (NH₄)₂SO₄, 10 mM KCl, 2 mM MgSO₄, 0.01% Triton-100, and 0.1 mg/ml bovine serum albumin, 10 pmol each primer, 0.01 μg of the pHBM905A plasmid, 0.2 mM each dNTPs. The cycling protocol was 95°C for 2 min; 25 cycles of 95°C for 10 s, 56°C for 15 s and 72°C for 30 s (A) or for 40 s (B) or for 50 s (C).

https://doi.org/10.1371/journal.pone.0131757.g005

Discussion

PCR is a standard industry process that is commonly used to produce linear DNA due to its significant facility and higher efficiency compared to bacterial fermentation. The applications of PCR products include DNA vaccines, RNA delivery, and gene therapy [24–28]. However, DNA polymerase is a costly component of PCR systems and can restrict the use of PCR.

Vandalia Research Company is capable of providing milligram or gram quantities of PCR-amplified DNA products using the Triathlon DNA method [29]. However, the expense of the DNA polymerase prevents the production of large quantities of DNA instantly for clinical consumption, particularly for industrial applications. Therefore, we sought an easy, inexpensive and rapid method to obtain a high-efficiency DNA polymerase.

Most DNA polymerases are expressed in E. coli. However, the low expression level and cumbersome purification protocol [1–3] prevents the rapid production of large quantities of DNA polymerase. As an inexpensive and effective strategy to obtain DNA polymerase, we expressed DNA polymerase in the yeast P. pastoris, which is frequently used to express and secret proteins. This yeast expression system has several advantages, including the availability of the strong methanol-induced alcohol oxidase (AOX1) promoter, the high expression level, and the convenient purification procedure. Moreover, P. pastoris can accommodate high cell densities and post-translational modifications of foreign proteins [30–32].

In this study, we constructed a recombinant P. pastoris strain expressing the DNA polymerase designated RKOD. We obtained approximately 250 mg of this recombinant DNA polymerase per liter, with an activity of approximately 19,384 U/mg. This study is the first to report the high expression of a DNA polymerase in P. pastoris. Evaluation of the characteristics of the RKOD DNA polymerase revealed that RKOD had excellent elongation rates, fidelity and thermostability, consistent with a previous study [33]. These results suggest that the RKOD DNA polymerase is suitable for long, accurate, and time-saving PCR [34–36]. We plan to test the ability of the RKOD DNA polymerase in a large-scale PCR of 200 milliliters or more. Preliminary results are promising (data not shown). Our results will contribute to the expansion of the applications of PCR. The successful expression of the RKOD DNA polymerase in a yeast system will reduce the cost of DNA polymerase production, shorten the procedure, and simplify the purification. This process serves as an excellent foundation for obtaining a large amount of high-fidelity DNA polymerase with good thermostability. The results of this study will also expand the applications of RKOD. For example, we can establish a large-scale PCR for producing linear DNA in response to new outbreaks of infectious disease. The successful expression of the recombinant DNA polymerase RKOD in the P. pastoris system will increase the advantages of the use of PCR in research.

Supporting Information

S1 Table. Primers used for PCR in this study

https://doi.org/10.1371/journal.pone.0131757.s001

(DOCX)

Acknowledgments

We thank Jun Yin for her collaboration in polymerase unit definitions. We thank Zhezhe Li for assistance in compiling the dataset.

Author Contributions

Conceived and designed the experiments: LM XY FW. Performed the experiments: FW SL HZ LB. Analyzed the data: FW LC ZZ XZ. Wrote the paper: FW.

References

1. Kim DJ, Pyun YR. Cloning, Expression, and Characterization of Thermostable DNA Polymerase from Thermoanaerobacter yonseiensis. Biochemistry and Molecurar Biology Reports. 2002; 35(3), 320–329.
- View Article
- Google Scholar
2. Dabrowski S, Kur J. Cloning and expression in Escherichia coli of the recombinant his-tagged DNA polymerases from Pyrococcus furiosus and Pyrococcus woesei. Protein expression and purification. 1998; 14(1), 131. pmid:9758761
- View Article
- PubMed/NCBI
- Google Scholar
3. Engelke DR, Krikos A, Bruck ME, Ginsburg D. Purification of Thermus aquaticus DNA polymerase expressed in Escherichia coli. Analytical biochemistry. 1990; 191(2), 396. pmid:2085185
- View Article
- PubMed/NCBI
- Google Scholar
4. Mroczkowski BS, Huvar A, Lernhardt W, Misono K, Nielson K, Scott B. Secretion of thermostable DNA polymerase using a novel baculovirus vector. Journal of Biological Chemistry. 1994; 269(18), 13522–13528. pmid:8175786
- View Article
- PubMed/NCBI
- Google Scholar
5. Pavlov AR, Belova GI, Kozyavkin SA, Slesarev AI. Helix–hairpin–helix motifs confer salt resistance and processivity on chimeric DNA polymerases. Proceedings of the National Academy of Sciences. 2002; 99(21), 13510–13515.
- View Article
- Google Scholar
6. Ahmad M, Hirz M, Pichler H, Schwab H. Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production. Applied microbiology and biotechnology. 2014; 98(12), 5301–5317. pmid:24743983
- View Article
- PubMed/NCBI
- Google Scholar
7. Wang Y, Prosen DE, Mei L, Sullivan JC, Finney M, Vander Horn PB. A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro. Nucleic acids research. 2004; 32(3), 1197–1207. pmid:14973201
- View Article
- PubMed/NCBI
- Google Scholar
8. Zhang GM, Hu Y, Zhuang YH, Ma LX, Zhang XE. Molecular cloning and heterologous expression of an alkaline xylanase from Bacillus pumilus HBP8 in Pichia pastoris. Biocatalysis and Biotransformation. 2006; 24(5), 371–379.
- View Article
- Google Scholar
9. Wu Q, Zhong X, Zhai C, Yang J, Chen X, Chen L, et al. A series of novel directional cloning and expression vectors for blunt-end ligation of PCR products. Biotechnology letters. 2010; 32(3), 439–443. pmid:19915798
- View Article
- PubMed/NCBI
- Google Scholar
10. Sambrook J, Russell DW, Russell DW. Molecular cloning: a laboratory manual (3-volume set). Cold Spring Harbor, New York: Cold spring harbor laboratory press. 2001.
11. Chen GQ, Choi I, Ramachandran B, Gouaux JE. Total gene synthesis: novel single-step and convergent strategies applied to the construction of a 779 base pair bacteriorhodopsin gene. Journal of the American Chemical Society. 1994; 116(19), 8799–8800.
- View Article
- Google Scholar
12. Wang F, Wang X, Yu X, Fu L, Liu Y, Ma L, et al. High-Level Expression of Endo-β-N-Acetylglucosaminidase H from Streptomyces plicatus in Pichia pastoris and Its Application for the Deglycosylation of Glycoproteins. PLoS ONE. 2015; 10(3): e0120458. pmid:25781897
- View Article
- PubMed/NCBI
- Google Scholar
13. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227(5259), 680–685. pmid:5432063
- View Article
- PubMed/NCBI
- Google Scholar
14. Grippo P, Richardson CC. Deoxyribonucleic acid polymerase of bacteriophage T7. Journal of Biological Chemistry. 1971; 246(22), 6867–6873. pmid:4942327
- View Article
- PubMed/NCBI
- Google Scholar
15. Lundberg K, Shoemaker D, Adams M, Short J, Sorge J, Mathur E. High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus. Gene. 1991; 108(1), 1–6. pmid:1761218
- View Article
- PubMed/NCBI
- Google Scholar
16. Rao B, Zhong X, Wang Y, Wu Q, Jiang Z, Ma L. Efficient vectors for expression cloning of large numbers of PCR fragments in P. pastoris. Yeast. 2010; 27(5), 285–292. pmid:20148389
- View Article
- PubMed/NCBI
- Google Scholar
17. Silver J, Keerikatte V. Novel use of polymerase chain reaction to amplify cellular DNA adjacent to an integrated provirus. Journal of virology. 1989; 63(5), 1924–1928. pmid:2704070
- View Article
- PubMed/NCBI
- Google Scholar
18. McCleary BV. A simple assay procedure for β-D-mannanase. Carbohydrate research. 1978; 67(1), 213–221. pmid:213193
- View Article
- PubMed/NCBI
- Google Scholar
19. Maley F, Trimble RB, Tarentino AL, Plummer TH. Characterization of glycoproteins and their associated oligosaccharides through the use of endoglycosidases. Analytical biochemistry. 1989; 180(2), 195–204. pmid:2510544
- View Article
- PubMed/NCBI
- Google Scholar
20. Muramatsu T. Demonstration of an endo-glycosidase acting on a glycoprotein. Journal of Biological Chemistry. 1971; 246(17), 5535–5537. pmid:4108054
- View Article
- PubMed/NCBI
- Google Scholar
21. Clark SE, Muslin EH, Henson CA. Effect of adding and removing N‐glycosylation recognition sites on the thermostability of barley α‐glucosidase. Protein Engineering Design and Selection. 2004; 17(3), 245–249.
- View Article
- Google Scholar
22. Somera AF, Pereira MG, Guimarães LHS, Polizeli MLTM, Terenzi HC, Furriel RPM, et al. Effect of glycosylation on the biochemical properties of β-xylosidases from Aspergillus versicolor. The journal of microbiology. 2009; 47(3), 270–276. pmid:19557344
- View Article
- PubMed/NCBI
- Google Scholar
23. Li M, Lin YC, Wu CC, Liu HS. Enhancing the efficiency of a PCR using gold nanoparticles. Nucleic acids research. 2005; 33(21), e184–e184. pmid:16314298
- View Article
- PubMed/NCBI
- Google Scholar
24. Shen X, Söderholm J, Lin F, Kobinger G, Bello A, Gregg DA, et al. Influenza A vaccines using linear expression cassettes delivered via electroporation afford full protection against challenge in a mouse model. Vaccine. 2012; 30(48), 6946. pmid:22406460
- View Article
- PubMed/NCBI
- Google Scholar
25. Chattopadhyay S, Ely A, Bloom K, Weinberg MS, Arbuthnot P. Inhibition of hepatitis B virus replication with linear DNA sequences expressing antiviral micro-RNA shuttles. Biochemical and biophysical research communications.2009; 389(3), 484–489. pmid:19733548
- View Article
- PubMed/NCBI
- Google Scholar
26. Chen ZY, Yant SR, He CY, Meuse L, Shen S, Kay MA. Linear DNAs concatemerize in vivo and result in sustained transgene expression in mouse liver. Molecular Therapy. 2001; 3(3), 403–410. pmid:11273783
- View Article
- PubMed/NCBI
- Google Scholar
27. Murphy MB, Fuller ST, Richardson PM, Doyle SA. An improved method for the in vitro evolution of aptamers and applications in protein detection and purification. Nucleic acids research. 2003; 31(18), e110–e110. pmid:12954786
- View Article
- PubMed/NCBI
- Google Scholar
28. Musheev MU, Krylov SN. Selection of aptamers by systematic evolution of ligands by exponential enrichment: addressing the polymerase chain reaction issue. Analytica chimica acta. 2006; 564(1), 91–96. pmid:17723366
- View Article
- PubMed/NCBI
- Google Scholar
29. http://www.vandaliaresearch.com/triathlon_applications.php
30. Böer E, Steinborn G, Kunze G, Gellissen G. Yeast expression platforms. Applied microbiology and biotechnology. 2007; 77(3), 513–523. pmid:17924105
- View Article
- PubMed/NCBI
- Google Scholar
31. Jahic M, Veide A, Charoenrat T, Teeri T, Enfors SO. Process technology for production and recovery of heterologous proteins with Pichia pastoris. Biotechnology progress. 2006; 22(6), 1465–1473. pmid:17137292
- View Article
- PubMed/NCBI
- Google Scholar
32. Cereghino JL, Cregg JM. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS microbiology reviews. 2000; 24(1), 45–66. pmid:10640598
- View Article
- PubMed/NCBI
- Google Scholar
33. Takagi M, Nishioka M, Kakihara H, Kitabayashi M, Inoue H, Kawakami B, et al. Characterization of DNA polymerase from Pyrococcus sp. strain KOD1 and its application to PCR. Applied and environmental microbiology. 1997; 63(11), 4504–4510. pmid:9361436
- View Article
- PubMed/NCBI
- Google Scholar
34. Fujiwara S, Takagi M, Manaka T. Archaeon Pyrococcus kodakaraensis KOD1: application and evolution. Biotechnology annual review. 1998; 4, 259. pmid:9890143
- View Article
- PubMed/NCBI
- Google Scholar
35. Nishioka M, Mizuguchi H, Fujiwara S, Komatsubara S, Kitabayashi M, Uemura H, et al. Long and accurate PCR with a mixture of KOD DNA polymerase and its exonuclease deficient mutant enzyme. Journal of biotechnology. 2001; 88(2). 141–149. pmid:11403848
- View Article
- PubMed/NCBI
- Google Scholar
36. Benson LM, Null AP, Muddiman DC. Advantages of Thermococcus kodakaraenis (KOD) DNA polymerase for PCR-mass spectrometry based analyses. Journal of the American Society for Mass Spectrometry. 2003; 14(6), 601–604. pmid:12781461
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. Kim DJ, Pyun YR. Cloning, Expression, and Characterization of Thermostable DNA Polymerase from Thermoanaerobacter yonseiensis. Biochemistry and Molecurar Biology Reports. 2002; 35(3), 320–329.
View Article
Google Scholar

[2] View Article

[3] Google Scholar

[ref2] 2. Dabrowski S, Kur J. Cloning and expression in Escherichia coli of the recombinant his-tagged DNA polymerases from Pyrococcus furiosus and Pyrococcus woesei. Protein expression and purification. 1998; 14(1), 131. pmid:9758761
View Article
PubMed/NCBI
Google Scholar

[5] View Article

[6] PubMed/NCBI

[7] Google Scholar

[ref3] 3. Engelke DR, Krikos A, Bruck ME, Ginsburg D. Purification of Thermus aquaticus DNA polymerase expressed in Escherichia coli. Analytical biochemistry. 1990; 191(2), 396. pmid:2085185
View Article
PubMed/NCBI
Google Scholar

[9] View Article

[10] PubMed/NCBI

[11] Google Scholar

[ref4] 4. Mroczkowski BS, Huvar A, Lernhardt W, Misono K, Nielson K, Scott B. Secretion of thermostable DNA polymerase using a novel baculovirus vector. Journal of Biological Chemistry. 1994; 269(18), 13522–13528. pmid:8175786
View Article
PubMed/NCBI
Google Scholar

[13] View Article

[14] PubMed/NCBI

[15] Google Scholar

[ref5] 5. Pavlov AR, Belova GI, Kozyavkin SA, Slesarev AI. Helix–hairpin–helix motifs confer salt resistance and processivity on chimeric DNA polymerases. Proceedings of the National Academy of Sciences. 2002; 99(21), 13510–13515.
View Article
Google Scholar

[17] View Article

[18] Google Scholar

[ref6] 6. Ahmad M, Hirz M, Pichler H, Schwab H. Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production. Applied microbiology and biotechnology. 2014; 98(12), 5301–5317. pmid:24743983
View Article
PubMed/NCBI
Google Scholar

[20] View Article

[21] PubMed/NCBI

[22] Google Scholar

[ref7] 7. Wang Y, Prosen DE, Mei L, Sullivan JC, Finney M, Vander Horn PB. A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro. Nucleic acids research. 2004; 32(3), 1197–1207. pmid:14973201
View Article
PubMed/NCBI
Google Scholar

[24] View Article

[25] PubMed/NCBI

[26] Google Scholar

[ref8] 8. Zhang GM, Hu Y, Zhuang YH, Ma LX, Zhang XE. Molecular cloning and heterologous expression of an alkaline xylanase from Bacillus pumilus HBP8 in Pichia pastoris. Biocatalysis and Biotransformation. 2006; 24(5), 371–379.
View Article
Google Scholar

[28] View Article

[29] Google Scholar

[ref9] 9. Wu Q, Zhong X, Zhai C, Yang J, Chen X, Chen L, et al. A series of novel directional cloning and expression vectors for blunt-end ligation of PCR products. Biotechnology letters. 2010; 32(3), 439–443. pmid:19915798
View Article
PubMed/NCBI
Google Scholar

[31] View Article

[32] PubMed/NCBI

[33] Google Scholar

[ref10] 10. Sambrook J, Russell DW, Russell DW. Molecular cloning: a laboratory manual (3-volume set). Cold Spring Harbor, New York: Cold spring harbor laboratory press. 2001.

[ref11] 11. Chen GQ, Choi I, Ramachandran B, Gouaux JE. Total gene synthesis: novel single-step and convergent strategies applied to the construction of a 779 base pair bacteriorhodopsin gene. Journal of the American Chemical Society. 1994; 116(19), 8799–8800.
View Article
Google Scholar

[36] View Article

[37] Google Scholar

[ref12] 12. Wang F, Wang X, Yu X, Fu L, Liu Y, Ma L, et al. High-Level Expression of Endo-β-N-Acetylglucosaminidase H from Streptomyces plicatus in Pichia pastoris and Its Application for the Deglycosylation of Glycoproteins. PLoS ONE. 2015; 10(3): e0120458. pmid:25781897
View Article
PubMed/NCBI
Google Scholar

[39] View Article

[40] PubMed/NCBI

[41] Google Scholar

[ref13] 13. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227(5259), 680–685. pmid:5432063
View Article
PubMed/NCBI
Google Scholar

[43] View Article

[44] PubMed/NCBI

[45] Google Scholar

[ref14] 14. Grippo P, Richardson CC. Deoxyribonucleic acid polymerase of bacteriophage T7. Journal of Biological Chemistry. 1971; 246(22), 6867–6873. pmid:4942327
View Article
PubMed/NCBI
Google Scholar

[47] View Article

[48] PubMed/NCBI

[49] Google Scholar

[ref15] 15. Lundberg K, Shoemaker D, Adams M, Short J, Sorge J, Mathur E. High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus. Gene. 1991; 108(1), 1–6. pmid:1761218
View Article
PubMed/NCBI
Google Scholar

[51] View Article

[52] PubMed/NCBI

[53] Google Scholar

[ref16] 16. Rao B, Zhong X, Wang Y, Wu Q, Jiang Z, Ma L. Efficient vectors for expression cloning of large numbers of PCR fragments in P. pastoris. Yeast. 2010; 27(5), 285–292. pmid:20148389
View Article
PubMed/NCBI
Google Scholar

[55] View Article

[56] PubMed/NCBI

[57] Google Scholar

[ref17] 17. Silver J, Keerikatte V. Novel use of polymerase chain reaction to amplify cellular DNA adjacent to an integrated provirus. Journal of virology. 1989; 63(5), 1924–1928. pmid:2704070
View Article
PubMed/NCBI
Google Scholar

[59] View Article

[60] PubMed/NCBI

[61] Google Scholar

[ref18] 18. McCleary BV. A simple assay procedure for β-D-mannanase. Carbohydrate research. 1978; 67(1), 213–221. pmid:213193
View Article
PubMed/NCBI
Google Scholar

[63] View Article

[64] PubMed/NCBI

[65] Google Scholar

[ref19] 19. Maley F, Trimble RB, Tarentino AL, Plummer TH. Characterization of glycoproteins and their associated oligosaccharides through the use of endoglycosidases. Analytical biochemistry. 1989; 180(2), 195–204. pmid:2510544
View Article
PubMed/NCBI
Google Scholar

[67] View Article

[68] PubMed/NCBI

[69] Google Scholar

[ref20] 20. Muramatsu T. Demonstration of an endo-glycosidase acting on a glycoprotein. Journal of Biological Chemistry. 1971; 246(17), 5535–5537. pmid:4108054
View Article
PubMed/NCBI
Google Scholar

[71] View Article

[72] PubMed/NCBI

[73] Google Scholar

[ref21] 21. Clark SE, Muslin EH, Henson CA. Effect of adding and removing N‐glycosylation recognition sites on the thermostability of barley α‐glucosidase. Protein Engineering Design and Selection. 2004; 17(3), 245–249.
View Article
Google Scholar

[75] View Article

[76] Google Scholar

[ref22] 22. Somera AF, Pereira MG, Guimarães LHS, Polizeli MLTM, Terenzi HC, Furriel RPM, et al. Effect of glycosylation on the biochemical properties of β-xylosidases from Aspergillus versicolor. The journal of microbiology. 2009; 47(3), 270–276. pmid:19557344
View Article
PubMed/NCBI
Google Scholar

[78] View Article

[79] PubMed/NCBI

[80] Google Scholar

[ref23] 23. Li M, Lin YC, Wu CC, Liu HS. Enhancing the efficiency of a PCR using gold nanoparticles. Nucleic acids research. 2005; 33(21), e184–e184. pmid:16314298
View Article
PubMed/NCBI
Google Scholar

[82] View Article

[83] PubMed/NCBI

[84] Google Scholar

[ref24] 24. Shen X, Söderholm J, Lin F, Kobinger G, Bello A, Gregg DA, et al. Influenza A vaccines using linear expression cassettes delivered via electroporation afford full protection against challenge in a mouse model. Vaccine. 2012; 30(48), 6946. pmid:22406460
View Article
PubMed/NCBI
Google Scholar

[86] View Article

[87] PubMed/NCBI

[88] Google Scholar

[ref25] 25. Chattopadhyay S, Ely A, Bloom K, Weinberg MS, Arbuthnot P. Inhibition of hepatitis B virus replication with linear DNA sequences expressing antiviral micro-RNA shuttles. Biochemical and biophysical research communications.2009; 389(3), 484–489. pmid:19733548
View Article
PubMed/NCBI
Google Scholar

[90] View Article

[91] PubMed/NCBI

[92] Google Scholar

[ref26] 26. Chen ZY, Yant SR, He CY, Meuse L, Shen S, Kay MA. Linear DNAs concatemerize in vivo and result in sustained transgene expression in mouse liver. Molecular Therapy. 2001; 3(3), 403–410. pmid:11273783
View Article
PubMed/NCBI
Google Scholar

[94] View Article

[95] PubMed/NCBI

[96] Google Scholar

[ref27] 27. Murphy MB, Fuller ST, Richardson PM, Doyle SA. An improved method for the in vitro evolution of aptamers and applications in protein detection and purification. Nucleic acids research. 2003; 31(18), e110–e110. pmid:12954786
View Article
PubMed/NCBI
Google Scholar

[98] View Article

[99] PubMed/NCBI

[100] Google Scholar

[ref28] 28. Musheev MU, Krylov SN. Selection of aptamers by systematic evolution of ligands by exponential enrichment: addressing the polymerase chain reaction issue. Analytica chimica acta. 2006; 564(1), 91–96. pmid:17723366
View Article
PubMed/NCBI
Google Scholar

[102] View Article

[103] PubMed/NCBI

[104] Google Scholar

[ref29] 29. http://www.vandaliaresearch.com/triathlon_applications.php

[ref30] 30. Böer E, Steinborn G, Kunze G, Gellissen G. Yeast expression platforms. Applied microbiology and biotechnology. 2007; 77(3), 513–523. pmid:17924105
View Article
PubMed/NCBI
Google Scholar

[107] View Article

[108] PubMed/NCBI

[109] Google Scholar

[ref31] 31. Jahic M, Veide A, Charoenrat T, Teeri T, Enfors SO. Process technology for production and recovery of heterologous proteins with Pichia pastoris. Biotechnology progress. 2006; 22(6), 1465–1473. pmid:17137292
View Article
PubMed/NCBI
Google Scholar

[111] View Article

[112] PubMed/NCBI

[113] Google Scholar

[ref32] 32. Cereghino JL, Cregg JM. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS microbiology reviews. 2000; 24(1), 45–66. pmid:10640598
View Article
PubMed/NCBI
Google Scholar

[115] View Article

[116] PubMed/NCBI

[117] Google Scholar

[ref33] 33. Takagi M, Nishioka M, Kakihara H, Kitabayashi M, Inoue H, Kawakami B, et al. Characterization of DNA polymerase from Pyrococcus sp. strain KOD1 and its application to PCR. Applied and environmental microbiology. 1997; 63(11), 4504–4510. pmid:9361436
View Article
PubMed/NCBI
Google Scholar

[119] View Article

[120] PubMed/NCBI

[121] Google Scholar

[ref34] 34. Fujiwara S, Takagi M, Manaka T. Archaeon Pyrococcus kodakaraensis KOD1: application and evolution. Biotechnology annual review. 1998; 4, 259. pmid:9890143
View Article
PubMed/NCBI
Google Scholar

[123] View Article

[124] PubMed/NCBI

[125] Google Scholar

[ref35] 35. Nishioka M, Mizuguchi H, Fujiwara S, Komatsubara S, Kitabayashi M, Uemura H, et al. Long and accurate PCR with a mixture of KOD DNA polymerase and its exonuclease deficient mutant enzyme. Journal of biotechnology. 2001; 88(2). 141–149. pmid:11403848
View Article
PubMed/NCBI
Google Scholar

[127] View Article

[128] PubMed/NCBI

[129] Google Scholar

[ref36] 36. Benson LM, Null AP, Muddiman DC. Advantages of Thermococcus kodakaraenis (KOD) DNA polymerase for PCR-mass spectrometry based analyses. Journal of the American Society for Mass Spectrometry. 2003; 14(6), 601–604. pmid:12781461
View Article
PubMed/NCBI
Google Scholar

[131] View Article

[132] PubMed/NCBI

[133] Google Scholar

Figures

Abstract

Introduction

Materials and Methods

Strains, vectors, media and reagents

Design and synthesis of the KOD and Sso7d genes

Constructing the expression vector for KOD expression in Pichia pastoris GS115 and E. coli

Expression and purification of RKOD in Pichia pastoris

Expression and purification of KOD and KOD-Sso in E. coli

DNA polymerase assays

Staining Glycoproteins in SDS-Polyacrylamide Gels

Thermostability analysis

Fidelity of DNA polymerase

PCR efficiency assay

Results

Optimization and synthesis of the RKOD gene

Expression and purification of the recombinant KOD DNA polymerase

Activity assessment of recombinant RKOD

Glycosylation analysis of RKOD

Temperature stability assay

PCR error rate of the RKOD DNA polymerase

PCR efficiency assay

Discussion

Supporting Information

S1 Table. Primers used for PCR in this study

Acknowledgments

Author Contributions

References