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‐ter‐minal 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 (PpHB) 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 Histag 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 chromo‐proteins showed an A660A280 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 (obs) of 35 s. The λ_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 PcjiB 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 Pfr 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 mUs, respectively). The formation of the Pfr 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‐terminus that in the His‐tagged form exhibits increased polarity.