Expression and Characterization of the RKOD DNA Polymerase in Pichia Pastoris

PLoS One. 2015 Jul 2;10(7):e0131757. doi: 10.1371/journal.pone.0131757. eCollection 2015.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Archaeal Proteins / biosynthesis
  • Archaeal Proteins / genetics*
  • Codon
  • DNA / chemistry
  • DNA-Binding Proteins / biosynthesis
  • DNA-Binding Proteins / genetics*
  • DNA-Directed DNA Polymerase / biosynthesis*
  • DNA-Directed DNA Polymerase / chemistry
  • DNA-Directed DNA Polymerase / genetics*
  • Escherichia coli / enzymology
  • Fungal Proteins / biosynthesis*
  • Fungal Proteins / genetics*
  • Glycoproteins / chemistry
  • Open Reading Frames
  • Pichia / enzymology*
  • Polymerase Chain Reaction
  • Protein Structure, Tertiary
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Sequence Analysis, DNA
  • Temperature
  • Thermococcus / enzymology

Substances

  • Archaeal Proteins
  • Codon
  • DNA-Binding Proteins
  • Fungal Proteins
  • Glycoproteins
  • Recombinant Proteins
  • Sso7d protein, Sulfolobus
  • DNA
  • DNA-Directed DNA Polymerase

Grant support

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.