Isolation of bacterial luciferases by affinity chromatography on 2,2-diphenylpropylamine-Sepharose: phosphate-mediated binding to an immobilized substrate analog

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Abstract
A covalently immobilized form of an inhibitor of bacterial luciferase, 2,2-diphenylpropylamine (D.vphi.PA), was an effective affinity resin for purifying this enzyme from several distinct bacterial species. The inhibitor is competitive with the luciferase aldehyde substrate but enhances binding of the flavin substrate FMNH2; comparable binding interactions occur with luciferase, the immobilized inhibitor D.vphi.PA-Sepharose, and the substrates. The effect of FMNH2 on the binding of luciferase to D.vphi.PA-Sepharose was mimicked by Pi; the luciferase-phosphate complex had a greater affinity for D.vphi.PA-Sepharose than did luciferase. This observation led to the development of a method using D.vphi.PA-Sepharose to purify bacterial luciferase. When crude enzyme in a high-phosphate buffer was applied to a column of the affinity matrix, the luciferase activity was removed from solution. After the column was washed with the same buffer to remove unbound protein, the luciferase was eluted with a non-phosphate cationic buffer. The affinity column has proven useful for rapid purification of luciferase in much greater yield than has been previously possible with standard ion-exchange techniques. This approach has allowed 1-step purification of luciferases from ammonium sulfate precipitates of Vibrio harveyi, Vibrio fischeri and Photobacterium phosphoreum. The dissociation constants in 0.10 M phosphate for the affinity ligand:luciferase complexes were 0.49, 0.28 and 0.15 .mu.M, respectively, for the 3 spp. The dissociation constant for the V. harveyi mutant AK-6, which has normal aldehyde binding but greatly reduced affinity for FMNH2, was 0.30 .mu.M, while that for the V. harveyi mutant AK-20, which has greatly reduced affinity for aldehyde but a slightly increased affinity for FMNH2, was 1.2 .mu.M. Preliminary experiments indicated that the yellow fluorescence protein (YFP) that participates, through energy transfer, in bioluminescent emission in V. fischeri strain Y-1 could be separated from the luciferase in this strain by chromatography on the affinity matrix, whereas other methods of separating luciferase and YFP have had limited success because of the binding of YFP to luciferase.