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

Fingerprint

Integrases
Bacteriophages
Serine
Genetic Recombination
Assays
Genes
Catalytic Domain
Gene therapy
Gene Transfer Techniques
Mutagenesis
Recombinases
Confocal microscopy
HEK293 Cells
Site-Directed Mutagenesis
Confocal Microscopy
Genetic Therapy
In Vitro Techniques
Fusion reactions
Genome
Amino Acids

Keywords

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

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Cell Biology
  • Molecular Biology

Cite this

In vivo and in vitro characterization of site-specific recombination of a novel serine integrase from the temperate phage EFC-1. / Yoon, Bohyun; Kim, Inki; Nam, Ja Ae; Chang, Hyo-Ihl; Ha, Chang Hoon.

In: Biochemical and Biophysical Research Communications, Vol. 473, No. 1, 22.04.2016, p. 336-341.

Research output: Contribution to journalArticle

@article{52e763c660f04b44a8a9bbebc279a110,
title = "In vivo and in vitro characterization of site-specific recombination of a novel serine integrase from the temperate phage EFC-1",
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.",
keywords = "Gene delivery system, Gene therapy, Integrase, Serine recombinase, Site-directed mutagenesis",
author = "Bohyun Yoon and Inki Kim and Nam, {Ja Ae} and Hyo-Ihl Chang and Ha, {Chang Hoon}",
year = "2016",
month = "4",
day = "22",
doi = "10.1016/j.bbrc.2016.03.106",
language = "English",
volume = "473",
pages = "336--341",
journal = "The BMJ",
issn = "0730-6512",
publisher = "Kluwer Academic Publishers",
number = "1",

}

TY - JOUR

T1 - In vivo and in vitro characterization of site-specific recombination of a novel serine integrase from the temperate phage EFC-1

AU - Yoon, Bohyun

AU - Kim, Inki

AU - Nam, Ja Ae

AU - Chang, Hyo-Ihl

AU - Ha, Chang Hoon

PY - 2016/4/22

Y1 - 2016/4/22

N2 - 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.

AB - 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.

KW - Gene delivery system

KW - Gene therapy

KW - Integrase

KW - Serine recombinase

KW - Site-directed mutagenesis

UR - http://www.scopus.com/inward/record.url?scp=84963642057&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84963642057&partnerID=8YFLogxK

U2 - 10.1016/j.bbrc.2016.03.106

DO - 10.1016/j.bbrc.2016.03.106

M3 - Article

VL - 473

SP - 336

EP - 341

JO - The BMJ

JF - The BMJ

SN - 0730-6512

IS - 1

ER -