Purification of bacterial luciferase native subunits and investigation of their folding, association and activity

Abstract

Bacterial luciferase from Vibrio harveyi is a heterodimeric enzyme comprised of two homologous subunits, α and β, encoded by adjacent genes, luxA and luxB. The level of accumulation of active enzyme was similar when the two subunits were expressed from a single transcript or separate transcripts. Cells carrying the individual plasmids accumulated large amounts of the individual subunits, but mixing the lysates or supernatants resulted in very low levels of active luciferase unless they were first denatured in urea followed by renaturation, which resulted in large amounts of active luciferase. This demonstrated that the productive folding of active luciferase is concerted and requires that partially folded subunits interact to form a complex. The α subunit produced by overproduction in Escherichia coli cells not encoding the β subunit was purified under native conditions to 98% purity. The α subunit was observed to have bioluminescence activity at the level of about 0.01% specific activity relative to the heterodimer. Gel filtration indicated the molecular weight was 62,000 kDa, possibly a homodimer (α[2]) Free α subunit has protease-labile regions similar to a in the heterodimer, but the active species is a resistant subpopulation. Concentration dependence of rates of a subunit refolding from urea to form the active species showed the reaction was higher than first-order. The β subunit was purified under native conditions to [greater than or equal to] 90% purity from ceils not encoding the α subunit, it was observed to have a bioluminescence specific activity higher than a, about 0.06% relative to the wild-type enzyme. The molecular weight from gel filtration (55,000 kDa) suggested it forms a homodimer (β[2]). The active form is a protease-labile subpopulation. Urea unfolding curves determined with intrinsic fluorescence emission measurements to compare the unfolding of luciferase and α in urea showed both proteins have at least one stable intermediate. In subunit mixing experiments, the highest activity was obtained from completely unfolding β subunit before mixing, but unfolding α did not make a significant difference. This indicates that in vivo, β must be maintained in a partially unfolded form before it encounters α to make a productive folding complex.