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

Fei Wang,Shuntang Li,Xing Zhong,Liang Chen,Lu Bian,Lixin Ma,Xiaolan Yu,Zhen Zhang,Hui Zhao,Eugene A Permyakov

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.

Highlights

More than 50 DNA polymerases have been cloned and sequenced from various organisms
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
For expression in P. pastoris, the KOD-Sso fragment was cloned into the pHBM905A vector fused with the MF-α leader sequence

Summary

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 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 [1,2,3]. The Pfu DNA polymerase was 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 PLOS ONE | DOI:10.1371/journal.pone.0131757. Baculovirus expression enables the PLOS ONE | DOI:10.1371/journal.pone.0131757 July 2, 2015

Methods

Results

Conclusion