title: Cloning, molecular characterization and expression of a DNA-ligase from a new bacteriophage: Phax1.
abstract: DNA ligases join 3' hydroxyl and 5' phosphate ends in double stranded DNA and are necessary for maintaining the integrity of genome. The gene encoding a new Escherichia phage (Phax1) DNA ligase was cloned and sequenced. The gene contains an open reading frame with 1,428 base pairs, encoding 475 amino acid residues. Alignment of the entire amino acid sequence showed that Phax1 DNA ligase has a high degree of sequence homology with ligases from Escherichia (vB_EcoM_CBA120), Salmonella (PhiSH19 and SFP10), Shigella (phiSboM-AG3), and Deftia (phiW-14) phages. The Phax1 DNA ligase gene was expressed under the control of the T7lac promoter on the pET-16b (+) in Escherichia coli Rossetta gami. The enzyme was then homogeneously purified by a metal affinity column. Enzymatic activity of the recombinant DNA ligase was assayed by an in-house PCR-based method.
authors.affiliation: Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Biotechnology Research Center, Tehran University of Medical Sciences, 1417614411, Tehran, Iran.
authors.forename: Neda
authors.initials: N
authors.lastname: Setayesh
authors.forename: Saleheh
authors.initials: S
authors.lastname: Sabouri-Shahrbabak
authors.forename: Hamid
authors.initials: H
authors.lastname: Bakherad
authors.forename: Zargham
authors.initials: Z
authors.lastname: Sepehrizadeh
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003090:Coliphages / Q000201:enzymology* / Q000235:genetics; D011088:DNA Ligases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004279:DNA, Viral; D004926:Escherichia coli / Q000235:genetics / Q000821:virology*; D008969:Molecular Sequence Data; D017900:Myoviridae / Q000201:enzymology* / Q000235:genetics; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D004279:DNA, Viral; D014764:Viral Proteins; D011088:DNA Ligases