EXPRESSION OF ACTIVE HUMAN DNA LIGASE-I IN ESCHERICHIA-COLI-CELLS THAT HARBOR A FULL-LENGTH DNA LIGASE-I CDNA CONSTRUCT

A recombinant plasmid for expression of full-length human DNA ligase I (phLig-I) was constructed in a plasmid/phage chimeric vector, pTD-T7N , which was derived from pUC118 by oligonucleotide-directed mutagenesi s. The insert contained a 2757-base pair coding sequence for a whole h uman DNA ligase I and an extra ACC codon adjacent to the ATG initiatio n codon. This ACC codon was required for achieving high levels of expr ession of full-length DNA ligase I in Escherichia coli strain BL21. Th e recombinant plasmid, which was designed to exploit the T7 late promo ter and the ATG initiation codon for beta-galactosidase was transfecte d into E. coli BL21 cells that express T7 RNA polymerase. The recombin ant clone produced relatively high levels of DNA ligase I with a molec ular mass of 130 kDa, as estimated by SDS-polyacrylamide gel electroph oresis. The DNA ligase was purified to near-homogeneity by the two-ste p column chromatographic procedure from BLphLig-I cells that had been induced with isopropyl beta-D-thiogalactoside. The specific activity, chromatographic behavior, kinetic properties, molecular mass, and anti genicity of the recombinant human DNA ligase I were indistinguishable from those of purified mammalian DNA ligase I. Metabolically labeling experiments with P-32(i) indicate that the recombinant DNA ligase I wa s present as an enzyme-AMP reaction intermediate, but not as a phospho protein, in the E. coli cells.