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• W2013216025 abstract "Abstract— 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 (PφB) and the unnatural chromophore precursors, phycocyanobilin (PCB) and phycoerythrobilin (PEB), produced covalent adducts that were similarly affinity purified. Both PφB and PCB adducts of ASPHYA-ST were photoactive-the PφB 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 photochem-ically 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 bilipro-tein 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." @default.
• W2013216025 created "2016-06-24" @default.
• W2013216025 creator A5028658969 @default.
• W2013216025 creator A5041543706 @default.
• W2013216025 date "1997-04-01" @default.
• W2013216025 modified "2023-09-25" @default.
• W2013216025 title "Purification and Characterization of Recombinant Affinity Peptide-Tagged Oat Phytochrome A" @default.
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• W2013216025 doi "https://doi.org/10.1111/j.1751-1097.1997.tb01920.x" @default.
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