Expression of phytochrome apoprotein from Avena sativa in Escherichia coli and formation of photoactive chromoproteins by assembly with phycocyanobilin

Abstract

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 "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-redhed 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, and P, absorption bands, which are blue-shifted relative to the corresponding bands of native phytochrome due to the particular phycocyanobilin structure. The 11 8-kDa fragment, only lacking the 6-kDa N-terminus, exhibits a strong P, band, but only a weak P, 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.

Influence of Expression System on Chromophore Binding and Preservation of Spectral Properties in Recombinant Phytochrome A

“…The PCR conditions for the synthesis of the recombinant 59-kDa and 39-kDa phytochrome apoproteins have been recently described (Hill et al, 1994). In brief, the plasmid pGP8.2-2 (Hershey et al, 1987) containing the AP3 gene of Avena sativa L. was used as PCR template.…”

“…Plasmids used for expression in bacteria (E. coli). The preparation of plasmids encoding the 45-kDa and 65-kDa apophytochrome fragments (pMEX45kD and pMEX65kD) was recently described (Hill et al, 1994). The plasmid which contains the cDNA encoding the 59-kDa fragment, was prepared by PCR using a forward primer, 5' GCGCATAGAATTCATGTGCGCAGTCATAGCCTACTTA-CAGC (ATG and first oat-sequence-specific codon GTC shown in bold), in combination with a reverse primer (R60), 5'…”

“…The fully grown cell cultures were harvested by centrifugation and either directly used, or stored at -70°C until needed. Cells were opened either by passage through a French press (Hill et al, 1994), or by employing an Ultra-Turrax (T25, Jahnke & Kunkel). For the latter procedure, the cell harvest pellet was suspended in a small volume of a sodium phosphate buffer (100 mM, pH 7.8, containing 2 mM EDTA, 2 mM dithiothreitol, and 2 mM phenylmethylsulfonyl fluoride), loaded into a syringe, and slowly dropped into the lysis vessel of the Ultra-Turrax which contained liquid nitrogen.…”

“…1-2 ml of a solution of recombinant apophytochrome was incubated with phycocyanobilin (final concentration 7-10 pM) at 4°C. Although chromopeptide formation could be observed already after a few minutes, the reconstitution was allowed to proceed for approximately 1 h. Redfar-red irradiation cycles using 667 nm interference and 730 nm cut-off filters were performed as described (Hill et al, 1994).…”

“…The reconstituted chromoproteins were compared, furthermore, with 65-kDa and 45-kDa fragments which correspond to the 59-kDa and 39-kDa peptides, respectively, exhibiting an intact complete N-terminus. The latter recombinant phytochrome fragments have recently been prepared by Hill et al (1994).…”