Engineering of DNA polymerase I from Thermus thermophilus using compartmentalized self-replication

Seaim Lwin Aye, Kei Fujiwara, Asuka Ueki, Nobuhide Doi

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Although compartmentalized self-replication (CSR) and compartmentalized partnered replication (CPR) are powerful tools for directed evolution of proteins and gene circuits, limitations remain in the emulsion PCR process with the wild-type Taq DNA polymerase used so far, including long run times, low amounts of product, and false negative results due to inhibitors. In this study, we developed a high-efficiency mutant of DNA polymerase I from Thermus thermophilus HB27 (Tth pol) suited for CSR and CPR. We modified the wild-type Tth pol by (i) deletion of the N-terminal 5′ to 3′ exonuclease domain, (ii) fusion with the DNA-binding protein Sso7d, (iii) introduction of four known effective point mutations from other DNA polymerase mutants, and (iv) codon optimization to reduce the GC content. Consequently, we obtained a mutant that provides higher product yields than the conventional Taq pol without decreased fidelity. Next, we performed four rounds of CSR selection with a randomly mutated library of this modified Tth pol and obtained mutants that provide higher product yields in fewer cycles of emulsion PCR than the parent Tth pol as well as the conventional Taq pol.

Original languageEnglish
Pages (from-to)170-176
Number of pages7
JournalBiochemical and Biophysical Research Communications
Volume499
Issue number2
DOIs
Publication statusPublished - 2018 May 5

Fingerprint

Thermus thermophilus
DNA Polymerase I
spleen exonuclease
Emulsions
Taq Polymerase
Polymerase Chain Reaction
Exonucleases
Gene Regulatory Networks
Base Composition
DNA-Binding Proteins
DNA-Directed DNA Polymerase
Point Mutation
Codon
Libraries
Fusion reactions
Networks (circuits)
Proteins

Keywords

  • Directed evolution
  • Emulsion PCR
  • Site-directed mutagenesis
  • Tth DNA polymerase

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Engineering of DNA polymerase I from Thermus thermophilus using compartmentalized self-replication. / Aye, Seaim Lwin; Fujiwara, Kei; Ueki, Asuka; Doi, Nobuhide.

In: Biochemical and Biophysical Research Communications, Vol. 499, No. 2, 05.05.2018, p. 170-176.

Research output: Contribution to journalArticle

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