Back to Search
Start Over
In vivo and in vitro characterization of site-specific recombination of a novel serine integrase from the temperate phage EFC-1
- Source :
- Biochemical and biophysical research communications. 473(1)
- Publication Year :
- 2016
-
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.
- Subjects :
- 0301 basic medicine
Genetic Vectors
Molecular Sequence Data
Biophysics
Mutagenesis (molecular biology technique)
Biochemistry
Polymerase Chain Reaction
Cell Line
Serine
03 medical and health sciences
Catalytic Domain
Recombinase
Humans
Bacteriophages
Site-specific recombination
Site-directed mutagenesis
Molecular Biology
Recombination, Genetic
Genome
Microscopy, Confocal
biology
Base Sequence
Integrases
Gene Transfer Techniques
Cell Biology
Genetic Therapy
Flow Cytometry
Molecular biology
Integrase
Temperateness
030104 developmental biology
HEK293 Cells
Microscopy, Fluorescence
Attachment Sites, Microbiological
Mutation
biology.protein
Mutagenesis, Site-Directed
Cre-Lox recombination
Plasmids
Subjects
Details
- ISSN :
- 10902104
- Volume :
- 473
- Issue :
- 1
- Database :
- OpenAIRE
- Journal :
- Biochemical and biophysical research communications
- Accession number :
- edsair.doi.dedup.....76a57af7bd79b7cffe955952cb235a63