Cloning, molecular characterization and expression of a DNA-ligase from a new bacteriophage: Phax1

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 amin...

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Bibliographic Details
Published in:World journal of microbiology & biotechnology 2013-12, Vol.29 (12), p.2227-2231
Main Authors: Setayesh, Neda, Sabouri-Shahrbabak, Saleheh, Bakherad, Hamid, Sepehrizadeh, Zargham
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Amino acids
Applied Microbiology
Binding sites
Biochemistry
Biomedical and Life Sciences
Biotechnology
Cloning
Cloning, Molecular
Coliphages - enzymology
Coliphages - genetics
DNA Ligases - chemistry
DNA Ligases - genetics
DNA Ligases - metabolism
DNA repair
DNA, Viral
E coli
Environmental Engineering/Biotechnology
Enzymatic activity
Enzyme kinetics
Enzymes
Escherichia coli
Escherichia coli - genetics
Escherichia coli - virology
Gene expression
Genetic testing
Genomes
Life Sciences
Microbiology
Molecular Sequence Data
Myoviridae - enzymology
Myoviridae - genetics
Original Paper
Phylogeny
Polymerase Chain Reaction
Protozoa
Salmonella
Sequence Alignment
Sequence Analysis, DNA
Sequence Homology, Amino Acid
Shigella
Studies
Viral Proteins - chemistry
Viral Proteins - genetics
Viral Proteins - metabolism
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Summary: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.
ISSN:0959-3993
1573-0972
DOI:10.1007/s11274-013-1386-1