Cloning, nucleotide sequence, and expression of the DNA ligase-encoding gene from Thermus filiformis

Abstract

The gene encoding Thermus filiformis (Tfi) DNA ligase was cloned and its nucleotide sequence was determined by the chain-termination method. The primary structure of Tfi DNA ligase was deduced from its nucleotide sequence. The Tfi DNA ligase comprises of 667 amino acid residues and its molecular mass was determined to be 75,936 Da. The deduced amino acid sequence of Tfi DNA ligase showed a 86.5% homology to Tth DNA ligase and 43.5% to E. coli DNA ligase. The Lys-116 of Lys-Val-Asp-Gly motif was proposed to be the active residue of Tfi DNA ligase. In comparison with the amino acid composition of DNA ligase, Tfi DNA ligase showed a significant increase in the proportion of charged residues, Arg and Glu, compared to E. coli DNA ligase. The G + C content in the first, second, and third positions of the codons used were 70.3%, 40.3%, and 90.3%, respectively. Codon usage in Tfi DNA ligase was heavily biased towards the use of G + C in the third position. Under tac promoter control, Tfi DNA ligase was overproduced to greater than 9% of E. coli BL26Blue cellular proteins.