EXPRESSION OF PHYTOCHROME APOPROTEIN FROM AVENA-SATIVA IN ESCHERICHIA-COLI AND FORMATION OF PHOTOACTIVE CHROMOPROTEINS BY ASSEMBLY WITH PHYCOCYANOBILIN

Phytochrome DNAs from oat (Avena sativa L.) encoding the full-length 1 24-kDa polypeptide, a 118-kDa fragment lacking the first 65 amino acid s, and two N-terninal fragments of 65 kDa and 45 kDa were subcloned an d expressed in Escherichia coli. Reducing the temperature to 25 degree s C during cell growth and the coexpression of chaperones improved the folding into a functional conformation for most of the polypeptides, and in one case the yield of polypeptides was also enhanced. A maximum yield of reconstitutable apoprotein was obtained by expressing the 65 -kDa fragment consisting of 595 amino acids. The apoproteins could be assembled in the Park with phycocyanobilin into photoreversible chromo proteins. The yield of photoreversible pigment could be further increa sed by far-red/red irradiation cycles, indicating that the presence of the chromophore promotes the correct folding of the binding site. The chromoproteins with an intact N-terminal domain exhibit P-r and P-tr absorption bands, which are blue-shifted relative to the corresponding bands of native phytochrome due to the particular phycocyanobilin str ucture. The 118-kDa fragment, only lacking the 6kDa N-terminus, exhibi ts a strong P-r band, but only a weak P-tr absorbance. This indicates an essential role of the front 6-kDa region of the protein in the form ation of the far-red absorbing chromophore-protein complex. Otherwise, the C-terminal region seems to be less important for photoreversibili ty as indicated by the function of the shorter fragments.