Improved Expression and Purification of the Carotenoid Biosynthetic Enzyme Z-ISO

Carotenoids are a large class of pigments that are essential for survival of plants and other species that consume these plant-derived compounds and their bioactive derivatives. The plant biosynthetic pathway is nuclear-encoded and localized in plastids. The pathway enzymes had been known for many years, except for a recently discovered isomerase, 15-cis-ζ-carotene isomerase (Z-ISO) which utilizes a novel mechanism to mediate isomerization in response to the redox state of its heme b cofactor. To further study this enzyme, a protocol is described which maximizes purification of a fusion between Maltose Binding Protein and Zea mays (maize) Z-ISO (MBP::Z-ISO) expressed in E. coli treated with heme biosynthesis precursors which were used to increase heme available for loading into the expressed protein. Further enrichment of the protein was accomplished by improved sonication to release membranes containing Z-ISO, an integral membrane protein, and collection of the membrane fraction which was subjected to Nickel affinity chromatography. The fusion protein bound to the column through a His-tag. The MBP::Z-ISO protein was released using histidine, and not imidazole which binds heme and would interfere with enzyme recovery. Purification of the 75.46 kD MBP::Z-ISO expressed in E. coli was accomplished with fivefold improvement of yield and doubled heme content compared to the previously published method Beltrán et al. (Nat Chem Biol 11(8):598-605, 2015). The newer protocol will yield, per liter of culture, 5-6 mg MBP::Z-ISO protein with ~1:1 heme to Z-ISO ratio.