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 (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.
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Claims 10
Claim · model-written summary
Full-length oat phytochrome A was purified to apparent homogeneity from yeast expression.
- Taxon
- Avena sativa (oat)
Avena sativae0 - Gene
- phytochrome A (ASPHYA)e0
- Outcome
- purified to apparent homogeneitye0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Full-length Avena sativa (oat) phytochrome A (ASPHYA) was expressed in the yeast Saccharomyces cerevisiae and purified to apparent homogeneity.”
Claim · model-written summary
Tagged ASPHYA yielded a 124 kDa polypeptide.
- Taxon
- oatAvena sativat0
- Outcome
- molecular masse0
- Value
- 124 kDae0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“produced a single polypeptide with a molecular mass of 124 kDa.”
t0“Oat”
Claim · model-written summary
ASPHYA-ST bound streptavidin agarose and eluted with 45% chromatographic efficiency.
- Taxon
- oatAvena sativat0
- Gene
- ASPHYA-STe0
- Outcome
- chromatographic efficiencye0
- Value
- 45%e0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“This strep-tagged polypeptide (ASPHYA-ST) bound tightly to streptavidin agarose and was selectively eluted using diaminobiotin, with a chromatographic efficiency of 45%.”
t0“Oat”
Claim · model-written summary
P phi B and PCB adducts were photoactive.
- Taxon
- oatAvena sativat0
- Gene
- ASPHYA-STe0
- Outcome
- photoactivee0
Not reportedDirection
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Both P phi B and PCB adducts of ASPHYA-ST were photoactive”
t0“Oat”
Claim · model-written summary
The P phi B adduct had properties nearly indistinguishable from native photoreceptor.
- Taxon
- oatAvena sativat0
- Outcome
- spectrophotometric propertiese0
- Direction
- nearly indistinguishablee0
- Comparator
- native photoreceptore0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“the P phi B adduct displaying spectrophotometric properties nearly indistinguishable from those of the native photoreceptor”
t0“Oat”
Claim · model-written summary
The PCB adduct had blue-shifted absorption maxima.
- Taxon
- oatAvena sativat0
- Outcome
- absorption maximae0
- Direction
- blue-shiftede0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“the PCB adduct exhibiting blue-shifted absorption maxima.”
t0“Oat”
Claim · model-written summary
The PEB adduct was photochemically inactive but fluorescent.
- Taxon
- oatAvena sativat0
- Outcome
- photochemically inactivee0
Not reportedDirection
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“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.”
t0“Oat”
Claim · model-written summary
PEB adduct excitation peaked at 576 nm.
- Taxon
- oatAvena sativat0
- Outcome
- excitation maximume0
- Value
- 576 nme0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“an excitation maximum at 576 nm”
t0“Oat”
Claim · model-written summary
PEB adduct emission peaked at 586 nm.
- Taxon
- oatAvena sativat0
- Outcome
- emission maximae0
- Value
- 586 nme0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“emission maxima at 586 nm”
t0“Oat”
Claim · model-written summary
ASPHYA-ST and its three bilin adducts were homodimers.
- Taxon
- oatAvena sativat0
- Gene
- ASPHYA-STe0
- Outcome
- homodimerse0
Not reportedDirection
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“ASPHYA-ST and its three bilin adducts were homodimers”
t0“Oat”