Recombinant Phycobiliproteins

Cai, Yuping; Murphy, John T.; Wedemayer, Gary J.; Glazer, Alexander N.

doi:10.1006/abio.2000.4979

Cited by 67 publications

(31 citation statements)

References 71 publications

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“…Extracts were clarified by centrifugation at 17,000 ϫ g for 20 min to pellet cell wall debris, inclusion bodies, and unbroken cells. HTApcA/ApcB was co-purified by metal affinity chromatography as described (47). The purified protein solution, which contained both subunits in a ϳ1:1 ratio as judged by SDS-PAGE, was dialyzed against 20 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM 2-mercaptoethanol, concentrated by ultrafiltration to ϳ1 mg/ml, and stored in aliquots at Ϫ20°C until required.…”

Section: Methodsmentioning

confidence: 99%

Identification and Characterization of a New Class of Bilin Lyase

Shen

Saunée

Williams

et al. 2006

Journal of Biological Chemistry

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Synechococcus sp. PCC 7002 and all other cyanobacteria that synthesize phycocyanin have a gene, cpcT, that is paralogous to cpeT, a gene of unknown function affecting phycoerythrin synthesis in Fremyella diplosiphon. A cpcT null mutant contains 40% less phycocyanin than wild type and produces smaller phycobilisomes with red-shifted absorbance and fluorescence emission maxima. Phycocyanin from the cpcT mutant has an absorbance maximum at 634 nm compared with 626 nm for the wild type. The phycocyanin ␤-subunit from the cpcT mutant has slightly smaller apparent molecular weight on SDS-PAGE. Purified phycocyanins from the cpcT mutant and wild type were cleaved with formic acid, and the products were analyzed by SDS-PAGE. No phycocyanobilin chromophore was bound to the peptide containing Cys-153 derived from the phycocyanin ␤-subunit of the cpcT mutant. Recombinant CpcT was used to perform in vitro bilin addition assays with apophycocyanin (CpcA/CpcB) and phycocyanobilin. Depending on the source of phycocyanobilin, reaction products with CpcT had absorbance maxima between 597 and 603 nm as compared with 638 nm for the control reactions, in which mesobiliverdin becomes covalently bound. After trypsin digestion and reverse phase high performance liquid chromatography, the CpcT reaction product produced one major phycocyanobilin-containing peptide. This peptide had a retention time identical to that of the tryptic peptide that includes phycocyanobilin-bound, cysteine 153 of wild-type phycocyanin. The results from characterization of the cpcT mutant as well as the in vitro biochemical assays demonstrate that CpcT is a new phycocyanobilin lyase that specifically attaches phycocyanobilin to Cys-153 of the phycocyanin ␤-subunit. Cyanobacteria and red algae contain peripheral, light-harvesting complexes called phycobilisomes (PBS).2 These macromolecular complexes are composed of two types of proteins: colored phycobiliproteins (PBPs), which absorb and transmit light energy to the photosynthetic reaction centers, and non-pigmented linker proteins, which direct the assembly of PBS (1). In many cyanobacteria, including Synechococcus sp. PCC 7002, PBS are composed of two blue-colored PBPs: phycocyanin (PC) and allophycocyanin (AP). These PBPs are composed of two subunits, ␣ and ␤ (2), which are members of the globin superfamily (3). The ␣-and ␤-subunits of PC and AP carry at least one linear tetrapyrrole chromophore (bilin) called phycocyanobilin (PCB), which is covalently attached to specific cysteine residues via thioether linkages (see supplemental Fig. 1) (4). The bilin chromophores of PBPs are derived from heme and are related to the phytochromobilin (5), the photoisomerizable chromophore of the plant protein, phytochrome (6). X-ray crystallographic analyses of PC have shown that the chiral C3 1 carbons of the PCB chromophores attached at cysteines ␣84 and ␤82 are in the R configuration, whereas the C3

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Section: Methodsmentioning

confidence: 99%

Identification and Characterization of a New Class of Bilin Lyase

Shen

Saunée

Williams

et al. 2006

Journal of Biological Chemistry

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“…As an approach to enhance the properties of phycobiliprotein biomarkers in fluorescence immunoassay, these phycobiliproteins were oligomerized to nano-complexes via affinity tags, such as the GCN4 leucine zipper domain: the resulting complexes showed significantly increased brightness. 19 …”

Section: Introductionmentioning

confidence: 99%

Orange fluorescent proteins constructed from cyanobacteriochromes chromophorylated with phycoerythrobilin

Sun

Tang

et al. 2014

Photochem Photobiol Sci

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Cyanobacteriochromes are a structurally and spectrally highly diverse class of phytochrome-related photosensory biliproteins. They contain one or more GAF domains that bind phycocyanobilin (PCB) autocatalytically; some of these proteins are also capable of further modifying PCB to phycoviolobilin or rubins. We tested the chromophorylation with the non-photochromic phycoerythrobilin (PEB) of 16 cyanobacteriochrome GAFs from Nostoc sp. PCC 7120, of Slr1393 from Synechocystis sp. PCC 6803, and of Tlr0911 from Thermosynechococcus elongatus BP-1. Nine GAFs could be autocatalytically chromophorylated in vivo/in E. coli with PEB, resulting in highly fluorescent biliproteins with brightness comparable to that of fluorescent proteins like GFP. In several GAFs, PEB was concomitantly converted to phycourobilin (PUB) during binding. This not only shifted the spectra, but also increased the Stokes shift.The chromophorylated GAFs could be oligomerized further by attaching a GCN4 leucine zipper domain, thereby enhancing the absorbance and fluorescence of the complexes. The presence of both PEB and PUB makes these oligomeric GAF-"bundles" interesting models for energy transfer akin to the antenna complexes found in cyanobacterial phycobilisomes. The thermal and photochemical stability and their strong brightness make these constructs promising orange fluorescent biomarkers.

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“…Deleterious UV-B rays damage protein, enzymes, uptake mechanisms, photosynthetic pigments and ultimately photosynthesis by dimer formation in genes Prasad et al, 2006). Cyanobacteria are the oldest, phylogenetically primitive group of photosynthetic prokaryotes of pre cambrian period (2.8-3.5x10 g yr) (Sinha and Hader, 1996a) and rich in photosynthetic pigments especially phycobilisomes (Cai et al, 2001). Spirulina fusiformis is an aseptate, filamentous, polymorphic, protein-rich microalgae (60-70%).It is rich in various useful biologically active compounds like phycobiliproteins (PBPs).…”

Section: Introductionmentioning

confidence: 99%

Biochemical and spectroscopic changes in phycobiliproteins of the protein-rich Cyanobacterium, Spirulina fusiformis induced by UV-B radiation

Prasad

2009

JANS

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The present study indicated that the Increase in UV-B exposure ( 312.67 nm, 2.5 Wm -2 ) to Spirulina fusiformis, a highly protein-rich (60-71%) helically coiled, unicellular, polymorphous cyanobacterium not only bleached phycocyanin (PC) but also decreased its content Absorption spectra of UV-B treated culture showed significant decline in the absorbance at the peak 620nm indicating the presence of PC. The absorbance decreased by increasing UV-B exposure. One hour simultaneous UV-B+PAR (Photosynthetic Active Radiation) exposure protected 33 percent damage of phycocyanin.

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Recombinant Phycobiliproteins

Cited by 67 publications

References 71 publications

Identification and Characterization of a New Class of Bilin Lyase

Identification and Characterization of a New Class of Bilin Lyase

Orange fluorescent proteins constructed from cyanobacteriochromes chromophorylated with phycoerythrobilin

Biochemical and spectroscopic changes in phycobiliproteins of the protein-rich Cyanobacterium, Spirulina fusiformis induced by UV-B radiation

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