Bacteriophage T7 DNA ligase. Overexpression, purification, crystallization, and characterization.
The bacteriophage T7 DNA ligase gene was amplified using polymerase chain reaction-based methods and cloned into a T7 promoter-based expression vector. The protein was overexpressed to greater than 15% of total soluble protein and purified to homogeneity, yielding 60-70 mg of protein per liter of bacterial culture. An initial physical and biochemical characterization of the enzyme reveals that it exists as a monomer and can ligate nicked, cohesive, and blunt-ended DNA fragments. Inhibition of the enzyme activity by a nonhydrolyzable ATP analogue was also investigated. The enzyme has been crystallized from methoxypolyethylene glycol. The crystals are of the orthorhombic space group P2(1)2(1)2 and diffract to 2.6 A. The unit cell dimensions are a = 66.1 A, b = 87.6 A, and c = 78.6 A, with one monomer in the asymmetric unit (Vm = 2.77 A3/Da). This is the first member of the DNA ligase family of enzymes to be crystallized.
Doherty
A J
AJ
Laboratory of Molecular Biophysics, University of Oxford, United Kingdom.
Ashford
S R
SR
Subramanya
H S
HS
Wigley
D B
DB
D017123:Bacteriophage T7 / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D011088:DNA Ligases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D017931:DNA Primers; D011994:Recombinant Proteins; D011088:DNA Ligases