Simple Isolation Method and Assay for T4 DNA Ligase and Characterization of the Purified Enzyme

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Abstract
A method for the isolation of [phage] T4-amber-N82-induced DNA ligase [from infected Escherichia coli] is described which results in a nearly homogeneous enzyme preparation after 2 column chromatographic steps. The enzyme is detected during the purification by its ability to form a stable acid-precipitable enzyme-adenylate complex. Some properties of the assay, such as the effect of salt, temperature and incubation time, are presented. The isolated enzyme and its adenylate complex are characterized by acrylamide gel electrophoresis under native and denaturing conditions, and by isoelectric focusing. The purified enzyme exhibits a MW of .apprx. 60,000. Isoelectric focusing yields at least 5 protein components, which are able to form an enzyme-adenylate complex. The main activity possesses a pI [isoelectric point] of 6. The enzyme preparation is capable of repairing [phage] T5+ DNA known to contain .apprx. 4 or 5 single-strand breaks, to circularize .lambda. DNA and to join HindIII and EcoRI fragments.