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.
authors.affiliation: Asan Institute for Life Sciences, Asan Medical Center, College of Medicine, University of Ulsan, 86 Asanbyeoungwon-gil, Songpa-gu, Seoul 138-736, Republic of Korea.
authors.forename: Bohyun
authors.initials: B
authors.lastname: Yoon
authors.affiliation: Asan Institute for Life Sciences, Asan Medical Center, College of Medicine, University of Ulsan, 86 Asanbyeoungwon-gil, Songpa-gu, Seoul 138-736, Republic of Korea.
authors.forename: Inki
authors.initials: I
authors.lastname: Kim
authors.affiliation: Asan Institute for Life Sciences, Asan Medical Center, College of Medicine, University of Ulsan, 86 Asanbyeoungwon-gil, Songpa-gu, Seoul 138-736, Republic of Korea.
authors.forename: Ja-Ae
authors.initials: JA
authors.lastname: Nam
authors.affiliation: College of Life Sciences & Biotechnology, Korea University, 5-1 Anam-dong, Seongbuk-gu, Seoul 136-701, Republic of Korea.
authors.forename: Hyo-Ihl
authors.initials: HI
authors.lastname: Chang
authors.affiliation: Asan Institute for Life Sciences, Asan Medical Center, College of Medicine, University of Ulsan, 86 Asanbyeoungwon-gil, Songpa-gu, Seoul 138-736, Republic of Korea. Electronic address: chhoonha@amc.seoul.kr.
authors.forename: Chang Hoon
authors.initials: CH
authors.lastname: Ha
mesh_terms: D001287:Attachment Sites, Microbiological; D001435:Bacteriophages / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D020134:Catalytic Domain; D002460:Cell Line; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D016678:Genome; D057809:HEK293 Cells; D006801:Humans; D019426:Integrases / Q000737:chemistry / Q000235:genetics*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D012694:Serine / Q000737:chemistry
chemical_list: D012694:Serine; D019426:Integrases
keywords: Gene delivery system; Gene therapy; Integrase; Serine recombinase; Site-directed mutagenesis