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 124-kDa polypeptide, a 118-kDa fragment lacking the first 65 amino acids, and two N-terminal fragments of 65 kDa and 45 kDa were subcloned and expressed in Escherichia coli. Reducing the temperature to 25 degrees 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 dark with phycocyanobilin into photoreversible chromoproteins. The yield of photoreversible pigment could be further increased 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 Pr and Pfr absorption bands, which are blue-shifted relative to the corresponding bands of native phytochrome due to the particular phycocyanobilin structure. The 118-kDa fragment, only lacking the 6-kDa N-terminus, exhibits a strong Pr band, but only a weak Pfr absorbance. This indicates an essential role of the front 6-kDa region of the protein in the formation of the far-red absorbing chromophore-protein complex. Otherwise, the C-terminal region seems to be less important for photoreversibility as indicated by the function of the shorter fragments.
Max-Planck-Institut für Strahlenchemic, Mülheim an der Ruhr, Germany.
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Hill
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Gärtner
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Towner
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Braslavsky
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Schaffner
D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D002523:Edible Grain / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D008969:Molecular Sequence Data; D052980:Phycobilins; D010798:Phycocyanin / Q000378:metabolism*; D010834:Phytochrome / Q000096:biosynthesis / Q000235:genetics*; D010957:Plasmids; D011758:Pyrroles / Q000378:metabolism*; D011994:Recombinant Proteins; D045725:Tetrapyrroles
D017931:DNA Primers; D052980:Phycobilins; D011758:Pyrroles; D011994:Recombinant Proteins; D045725:Tetrapyrroles; D010798:Phycocyanin; D010834:Phytochrome; C011069:phycocyanobilin