Purification and characterization of recombinant affinity peptide-tagged oat phytochrome A.
Full-length Avena sativa (oat) phytochrome A (ASPHYA) was expressed in the yeast Saccharomyces cerevisiae and purified to apparent homogeneity. Expression of an ASPHYA cDNA that encoded the full-length photoreceptor with a 15 amino acid 'strep-tag' peptide at its C-terminus produced a single polypeptide with a molecular mass of 124 kDa. This strep-tagged polypeptide (ASPHYA-ST) bound tightly to streptavidin agarose and was selectively eluted using diaminobiotin, with a chromatographic efficiency of 45%. Incubation of ASPHYA-ST with phytochromobilin (P phi B) and the unnatural chromophore precursors, phycocyanobilin (PCB) and phycoerythrobilin (PEB), produced covalent adducts that were similarly affinity purified. Both P phi B and PCB adducts of ASPHYA-ST were photoactive--the P phi B adduct displaying spectrophotometric properties nearly indistinguishable from those of the native photoreceptor, and the PCB adduct exhibiting blue-shifted absorption maxima. Although the PEB adduct of ASPHYA-ST was photochemically inactive, it was intensely fluorescent with an excitation maximum at 576 nm and emission maxima at 586 nm. The superimposability of its absorption and fluorescence excitation spectra established that a single biliprotein species was responsible for fluorescence from the adduct produced when ASPHYA-ST was incubated with PEB. Steric exclusion HPLC also confirmed that ASPHYA-ST and its three bilin adducts were homodimers, as has been established for phytochrome A isolated from natural sources. The ability to express and purify recombinant phytochromes with biochemical properties very similar to those of the native molecule should facilitate detailed structural analysis of this important class of photoreceptors.
Murphy
J T
JT
Section of Molecular and Cellular Biology, University of California, Davis 95616, USA.
Lagarias
J C
JC
D000595:Amino Acid Sequence; D018554:Avena / Q000737:chemistry*; D001483:Base Sequence; D004274:DNA, Recombinant; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010834:Phytochrome / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D051942:Phytochrome A; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013057:Spectrum Analysis
D004274:DNA, Recombinant; D010455:Peptides; D051942:Phytochrome A; D011994:Recombinant Proteins; D010834:Phytochrome