title: Influence of expression system on chromophore binding and preservation of spectral properties in recombinant phytochrome A.
abstract: N-Terminal deletion mutants of the plant photoreceptor phytochrome, additionally truncated at two different positions at their C-terminal ends, were expressed both in Escherichia coli and in yeast (Pichia pastoris) and converted into chromoproteins upon chromophore incorporation. The start and end positions of the cDNA employed (phyA from oat) mimic the positions of tryptic cleavage (deletion of the first 64 amino acids, and stop codons after amino acid positions 425 or 595, generating 39-kDa and 59-kDa peptides, respectively. The absorption properties and photochromicity upon red/far-red irradiation of these mutants were compared with their tryptic counterparts derived from native oat phytochrome and with recombinant products possessing intact N-termini, but C-terminal positions identical to those of the corresponding tryptic fragments (45-kDa and 65-kDa peptides). All recombinant 65-kDa and 59kDa peptides bound the chromophore after expression and showed the appropriate absorption spectra of the Pr and the Pfr forms. The smaller chromopeptides (45-kDa and 39-kDa) behaved differently depending on the expression system employed. E. coli-derived peptides exhibited a phytochrome-like difference spectrum only when the intact N-terminus was present (45-kDa product). The recombinant 39-kDa peptide from E. coli was incapable of chromophore binding whereas the identical peptide sequence expressed by P. pastoris formed a chromoprotein with phycocyanobilin. This recombinant phytochrome fragment exhibited a difference spectrum (Pr-Pfr) with an even larger Pfr absorption band than the comparable tryptic 39-kDa fragment. Selectivity of chromophore incorporation and spectral properties suggest that interactions between protein domains of phytochrome control the protein folding and the Pr/Pfr absorption characteristics. Evidently, trypsin digestion down to the 39-kDa fragment affects protein conformation also in terms of Pfr conservation.
authors.affiliation: Max-Planck-Institut für Strahlenchemie, Mülheim a.d. Ruhr, Germany.
authors.forename: W
authors.initials: W
authors.lastname: Gärtner
authors.forename: C
authors.initials: C
authors.lastname: Hill
authors.forename: K
authors.initials: K
authors.lastname: Worm
authors.forename: S E
authors.initials: SE
authors.lastname: Braslavsky
authors.forename: K
authors.initials: K
authors.lastname: Schaffner
mesh_terms: D018554:Avena / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D004926:Escherichia coli; D008027:Light; D008969:Molecular Sequence Data; D015388:Organelles / Q000378:metabolism*; D010834:Phytochrome / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D051942:Phytochrome A; D010843:Pichia; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D051942:Phytochrome A; D011994:Recombinant Proteins; D010834:Phytochrome