Cloning, overexpression and nucleotide sequence of a thermostable DNA ligase-encoding gene

Abstract

Thermostable DNA ligase has been harnessed for the detection of single-base genetic diseases using the ligase chain reaction [Barany, Proc. Natl. Acad. Sci. USA 88 (1991) 189-193]. The Thermus thermophilus (Tth) DNA ligase-encoding gene (ligT) was cloned in Escherichia coli by genetic complementation of a ligts 7 defect in an E. coli host. Nucleotide sequence analysis of the gene revealed a single chain of 676 amino acid residues with 47% identity to the E. coli ligase. Under phoA promoter control, Tth ligase was overproduced to greater than 10% of E. coli cellular proteins. Adenylated and deadenylated forms of the purified enzyme were distinguished by apparent molecular weights of 81 kDa and 78 kDa, respectively, after separation via sodium dodecyl sulfate-polyacrylamide-gel electrophoresis.