Chapter 4
Improved Expression and Purification of the Carotenoid
Biosynthetic Enzyme Z-ISO
Eleanore T. Wurtzel and Jesu ´ s Beltra ´ n
Abstract
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 Beltra ´n
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.
Key words Plant carotenoid biosynthesis, Heme, Isomerase, 15-cis-ζ-carotene, Membrane, E. coli,
Protein purification
1 Introduction
Carotenoids and their bioactive derivatives constitute a large class
of pigments that enable plants to capture photosynthetic light
energy, protect against high light damage and regulate plant
growth and development in a complex environment. Dietary
sources of plant carotenoids are important for human health and
species survival. With these manifold roles of carotenoids in mind, a
multitude of opportunities exist for metabolic engineering and
Manuel Rodrı ´guez-Concepcio ´ n and Ralf Welsch (eds.), Plant and Food Carotenoids: Methods and Protocols,
Methods in Molecular Biology, vol. 2083, https://doi.org/10.1007/978-1-4939-9952-1_4,
© Springer Science+Business Media, LLC, part of Springer Nature 2020
53
Improved Expression and Purification of the Carotenoid
Biosynthetic Enzyme Z-ISO
Eleanore T. Wurtzel and Jesu ´ s Beltra ´ n
Abstract
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 Beltra ´n
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.
Key words Plant carotenoid biosynthesis, Heme, Isomerase, 15-cis-ζ-carotene, Membrane, E. coli,
Protein purification
1 Introduction
Carotenoids and their bioactive derivatives constitute a large class
of pigments that enable plants to capture photosynthetic light
energy, protect against high light damage and regulate plant
growth and development in a complex environment. Dietary
sources of plant carotenoids are important for human health and
species survival. With these manifold roles of carotenoids in mind, a
multitude of opportunities exist for metabolic engineering and
Manuel Rodrı ´guez-Concepcio ´ n and Ralf Welsch (eds.), Plant and Food Carotenoids: Methods and Protocols,
Methods in Molecular Biology, vol. 2083, https://doi.org/10.1007/978-1-4939-9952-1_4,
© Springer Science+Business Media, LLC, part of Springer Nature 2020
53