Large‐scale Generation of Affinity‐purified Recombinant Phytochrome Chromopeptide

Abstract

Two different yeast expression systems, Pichia pastoris and Hansenula polymorpha, are compared for their capability to express in functional form the 65 kDa N-terminal portion of oat phytochrome A (phyA, spanning amino acids 1-595). The front half of phytochrome was selected for this investigation because it exhibits a greater stability than the full-length protein, and it harbors full spectroscopic and kinetic properties of phytochrome, allowing an exact proof of the functional integrity of the recombinant material. In the comparison between the two expression systems used, special emphasis was given to optimizing the yield of the expression and to improving the quality of the expressed material with respect to the proportion of functional protein. From identical volumes of cell culture, H. polymorpha synthesized between 8- and 10-fold more functional protein than P. pastoris. Following the observation by Wu and Lagarias (Proc. Natl. Acad. Sci. USA 93, 8989-8994, 1996) that P. pastoris endogenously produces the chromophore of phytochrome, phytochromobilin (P phi B) in significant amounts that leads to formation of spectrally active phytochrome during expression, the invention of an alternative high-yield expression system was strongly demanded. A His6-tag was attached to the C-terminus of the recombinant protein, which allows for a convenient and efficient purification and selects the full-length proteins over translationally truncated peptides. Fully reconstituted chromoproteins showed an A660/A280 ratio of > 1.2, indicating the high degree of reconstitutable apoprotein obtained by this procedure. The assembly between apoprotein and the chromophore phycocyanobilin when followed time-resolved yielded a time constant (tau obs) of 35 s. The lambda max values of the red-(Pr) and the far red-absorbing (Pfr) forms of phytochrome (665 and 729 nm) of the recombinant 65 kDa chromopeptide, reconstituted with P phi B are nearly identical to those of native full-length oat phytochrome. The kinetic parameters of the affinity-purified 65 kDa phytochrome chromoprotein for the Pr-->I700--> -->Ptr conversion are compared to those of the recombinant 65 kDa chromoprotein, lacking the His-tag and to wild-type oat phytochrome. Referring to wild-type phytochrome allows determination of whether the recombinant material has lost spectral properties during the purification procedure. The decay of the primary intermediate (I700) occurs with nearly the same time constant for the His-tagged chromoprotein and for the reference (110 and 90 microseconds, respectively). The formation of the Ptr form was fitted with three exponentials in both the His-tagged and the reference chromoprotein with the middle component being slightly smaller and the longest component being remarkably larger for the His-tagged protein (1.5, 10 and 300 ms) than for the reference (1.4, 18 and 96 ms). This selective slowing down of the long kinetic component in the millisecond time range may be indicative of stronger interactions between protein domains involving the C...

Phytochromes With Noncovalently Bound Chromophores: The Ability of Apophytochromes to Direct Tetrapyrrole Photoisomerization¶†

“…Incubation of the recombinant apoprotein of phyA‐65 with PCB is known to result in the covalent binding of the chromophore and in the photochemistry characteristic of phytochromes (λ max [P r ] 652 nm, λ max [P fr ] 718 nm [16]; see also Fig. 4, lower panel).…”

“…The 3′‐methoxy derivative was synthesized as recently described (13). The recombinant apoprotein of the His‐tagged, 65 kDa, N‐terminal fragment of phyA ([phyA‐65], spanning amino acids 1–595) was produced in the methylotrophic yeast Hansenula polymorpha and purified according to published protocols, including affinity chromatography (16). The recombinant apoprotein of CphB from Calothrix sp.…”

Light-induced chromophore activity and signal transduction in phytochromes observed by 13 C and 15 N magic-angle spinning NMR

“…The u-[ 13 C, 15 N]-PCB was prepared following published methods (27). Preparation of Cph1⌬2 and phyA65 apo-and holoproteins were performed as described (34,35). For the measurements of the Pr state, samples were irradiated with light filtered through a far-red cut-off filter (max ϭ 730 nm).…”

Exploring Chromophore-Binding Pocket: High-Resolution Solid-State 1H–13C Interfacial Correlation NMR Spectra with Windowed PMLG Scheme

“…Preparation of uniformly 13 C- and 15 N-labeled PCB chromophore, Cph1Δ2 apo- and holoprotein was described elsewhere [34–36]. For NMR measurements of Pr Cph1Δ2 phytochrome at 100% occupancy, the sample was pre-irradiated with light from an array of far-red light-emitting diodes (LEDs) ( λ max  ≈ 730 nm, 20 nm full-width at half-maximum (FWHM)) surrounding a 1-mm bore glass capillary syringe.…”