In vivo and in vitro characterization of site-specific recombination of a novel serine integrase from the temperate phage EFC-1

Bohyun Yoon, Inki Kim, Ja Ae Nam, Hyo-Ihl Chang, Chang Hoon Ha

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

EFC-1 integrase is a site-specific recombinase that belongs to the large family of serine recombinase. In previously study, we isolated the temperate phage EFC-1, and characterized its genomic sequence. Within its genome, Orf28 was predicted encode a 464 amino acid of a putative integrase gene. In this study, EFC-1 integrase was characterized in vitro and in vivo. In vitro assay was performed using purified His-tag fusion integrase. Also, to identify which serine is involved in the catalytic domain, we used site-directed mutagenesis and analyzed by a recombination assay in vitro. In vivo assay involved PCR and confocal microscopy in HEK293 cells, and determined the minimal lengths of attP and attB sites. According to our results, the EFC-1 integrase-mediated recombination was site-specific and unidirectional system in vitro and in vivo. Serine 21 of EFC-1 integrase plays a major role in the catalytic domain, and minimal sizes of attB and attP was defined 48 and 54 bp. Our finding may help develop a useful tool for gene therapy and gene delivery system.

Original languageEnglish
Pages (from-to)336-341
Number of pages6
JournalBiochemical and Biophysical Research Communications
Volume473
Issue number1
DOIs
Publication statusPublished - 2016 Apr 22

Keywords

  • Gene delivery system
  • Gene therapy
  • Integrase
  • Serine recombinase
  • Site-directed mutagenesis

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Cell Biology
  • Molecular Biology

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