LARGE-SCALE GENERATION OF AFFINITY-PURIFIED RECOMBINANT PHYTOCHROME CHROMOPEPTIDE

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, spanni ng amino acids 1-595). The front half of phytochrome was selected for this investigation because it exhibits a greater stability than the fu ll-length protein, and it harbors full spectroscopic and kinetic prope rties of phytochrome, allowing an exact proof of the functional integr ity of the recombinant material. In the comparison between the two exp ression systems used, special emphasis was given to optimizing the yie ld of the expression and to improving the quality of the expressed mat erial with respect to the proportion of functional protein. From ident ical volumes of cell culture, H. polymorpha synthesized between 8- and 10-fold more functional protein than P. pastoris. Following the obser vation by Wu and Lagarias (Proc. Natl. Acad. Sci. USA 93, 8989-8994, 1 996) that P. pastoris endogenously produces the chromophore of phytoch rome, phytochromobilin (P phi B) in significant amounts that leads to formation of spectrally active phytochrome during expression, the inve ntion of an alternative high-yield expression system was strongly dema nded. A His(6)-tag was attached to the C-terminus of the recombinant p rotein, which allows for a convenient and efficient purification and s elects the full-length proteins over translationally truncated peptide s. Fully reconstituted chromoproteins showed an A(660)/A(280) ratio of >1.2, indicating the high degree of reconstitutable apoprotein obtain ed by this procedure. The assembly between apoprotein and the chromoph ore phycocyanobilin when followed time-resolved yielded a time constan t (tau(obs)) of 35 s. The lambda(max) values of the red-(P-r) and the far red-absorbing (P-fr) forms of phytochrome (665 and 729 mn) of the recombinant 65 kDa chromopeptide, reconstituted with P phi B are nearl y identical to those of native full-length oat phytochrome. The kineti c parameters of the affinity-purified 65 kDa phytochrome chromoprotein for the P-r --> I-700 --> --> P-fr conversion are compared to those o f the recombinant 65 kDa chromoprotein, lacking the His-tag and to wil d-type oat phytochrome. Referring to wild-type phytochrome allows dete rmination of whether the recombinant material has lost spectral proper ties during the purification procedure. The decay of the primary inter mediate (I-700) occurs with nearly the same time constant for the His- tagged chromoprotein and for the reference (110 and 90 mu s, respectiv ely). The formation of the P-fr form was fitted with three exponential s in both the His-tagged and the reference chromoprotein with the midd le component being slightly smaller and the longest component being re markably larger for the His-tagged protein (1.5, 10 and 300 ms) than f or the reference (1.4, 18 and 96 ms). This selective slowing down of t he long kinetic component in the millisecond time range may be indicat ive of stronger interactions between protein domains involving the C-t erminus that in the His-tagged form exhibits increased polarity.