Purification and Reconstitution of PYP-phytochrome with Biliverdin and 4-hydroxycinnamic Acid

PYP-phytochrome (Ppr) is a unique photoreceptor that contains a blue light-absorbing photoactive yellow protein (PYP) domain, a red light-absorbing phytochrome domain, and a histidine kinase domain. This chapter describes overexpression of Ppr in a strain of Escherichia coli that allows covalent attachment of substoichiometric amounts of biliverdin in vivo. Ppr is then fully reconstituted with biliverdin, followed by attachment of 4-hydroxycinnamic acid (p-coumaric acid), in vitro. Holo-Ppr with both chromophores is then isolated via an affinity tag and quantified for chromophore attachment by analysis of the absorption spectrum for biliverdin and 4-hydroxycinnamic acid. We also provide conditions for measuring autophosphorylation of Ppr.