Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Using tissue cell culture to make a protein or polypeptide
Reexamination Certificate
2001-07-31
2004-05-25
Carlson, Karen Cochrane (Department: 1653)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Using tissue cell culture to make a protein or polypeptide
C435S070100, C435S325000, C435S252300, C435S252200, C435S455000, C435S468000, C435S449000, C435S419000, C435S007920, C435S320100, C435S006120
Reexamination Certificate
active
06740507
ABSTRACT:
INTRODUCTION
1. Field of the Invention
The field of the invention is recombinant cells engineered to express heterologous holo-phycobiliproteins.
2. Background of the Invention
The phycobiliproteins are a family of light-harvesting proteins found in cyanobacteria, red algae, and the cryptomonads. These proteins absorb strongly in the visible region of the spectrum because they carry various covalently attached linear tetrapyrrole prosthetic groups (bilins). Phycobiliproteins are tightly associated &agr;&bgr; heterodimers, in which each subunit carries bilin(s) thioether-linked to particular cysteinyl residues (1-3).
Steps involved in bilin biosynthesis and bilin addition to apophycobiliprotein subunits have been inferred from diverse studies (e.g., 4-6). However, the entire pathway from heme to a particular holophycobiliprotein subunit has not hitherto been reconstituted either in vitro or in vivo. This achievement is reported here.
The invention offers numerous applications in the enzymology and chemistry of phycobiliprotein synthesis, and enables the use of phycobiliproteins as in vivo fluorescent protein probes. Purified native phycobiliproteins and their subunits fluoresce strongly and, since 1982, have been widely used as external labels for cell sorting and analysis and a wide range of other fluorescence-based assays (7, 8). Fluorescent protein probes expressed in vivo, such as
Aequorea victoria
green fluorescent protein and its variants, have proved to be of great value in all fields of cell biology (9). Probes generated by the spontaneous addition of exogenously supplied phycoerythrobilin to recombinant apophytochrome fragments in living cells are also very promising (10). Phycobiliprotein constructs represent a broad array of spectroscopically distinctive proteins with photophysical properties superior to those of probes currently available, and phycobiliprotein subunits have proven highly versatile as fusion partners (11). The expression in prokaryotic or eukaryotic cells of genes encoding enzymes and apo-phycobiliprotein subunit-containing fusion proteins permits intracellular production of constructs carrying specific bilins at unique locations, with broad utility in addressing a variety of questions in cell biology.
SUMMARY OF THE INVENTION
The invention provides recombinant cells which express a fluorescent holo-phycobiliprotein fusion protein and methods of use. Cells which do not naturally express a fluorescent, holo-phycobiliprotein are genetically engineered to comprise a functional pathway for making a fluorescent, holo-phycobiliprotein. The holo-phycobiliprotein fusion proteins comprise a heterologous-to-the-cell, fluorescent, first holo-phycobiliprotein domain fused to a heterologous protein domain. In a particular embodiment, the cell makes and comprises components: a bilin, a recombinant bilin reductase, an apo-phycobiliprotein fusion protein precursor of the fusion protein comprising a corresponding apo-phycobiliprotein domain, and a recombinant phycobiliprotein domain-bilin lyase, which components react inside the cell to form the holo-phycobiliprotein fusion protein.
In a particular embodiment, the cell further comprises a heme and a heme oxygenase which react to form the bilin, particularly a recombinant heme oxygenase such as HO1.
In additional embodiments, the heterologous protein domain is fluorescent and spectroscopically distinguishable from the first holo-phycobiliprotein domain, the heterologous protein domain comprises a heterologous-to-the-cell, fluorescent, second holo-phycobiliprotein domain, the heterologous protein domain comprises a phytochrome domain, the heterologous protein domain comprises a green fluorescent protein (GFP) domain, and/or the fusion protein provides fluorescence resonance energy transfer between the first holo-phycobiliprotein domain and the heterologous protein domain.
The invention may be practiced in a wide variety of cells, including mammalian cells, yeast cells (e.g.
S. cerevisiae
), bacterial cells (e.g.
E. coli
), etc., which may be present in vitro, which are generally isolated from a host, or in situ.
In particularly exemplified applications, (a) the bilin is phycocyanobilin (PCB), the reductase is 3Z-phycocyanobilin:ferredoxin oxidoreductase (PcyA), the apo-phycobiliprotein domain is phycocyanin &agr; subunit domain, and the lyase is heterodimeric phycocyanin &agr; subunit phycocyanobilin lyase (CpcE and CpcF); (b) the bilin is phycocyanobilin (PCB), the reductase is 3Z-phycocyanobilin:ferredoxin oxidoreductase (PcyA), the apo-phycobiliprotein domain is phycoerythrocyanin apo-&agr; subunit domain, and the lyase is heterodimeric phycoerythrocyanin &agr; subunit phycoerythrocyanobilin lyase (PecE and PecF), which further catalyzes the isomerization of the bound bilin to phycobiliviolin; and (c) the bilin is phycoerythrobilin (PEB), the reductase is 3Z-phycoerythrobilin:ferredoxin oxidoreductase (PebA and PebB), the apo-phycobiliprotein domain is phycoerythrin apo-&agr; subunit domain, and the lyase is heterodimeric C-phycoerythrin &agr; subunit phycoerythrobilin lyase (CpeY and CpeZ).
The invention also provides methods of making and using the subject cells and fusion proteins. For example, subject methods include making holo-phycobiliprotein fusion proteins by growing the subject recombinant cells under conditions wherein the cells express the holo-phycobiliprotein fusion protein, which methods may further comprise the step of isolating the holo-phycobiliprotein fusion protein, and/or the step of specifically detecting, including detecting the location, movement, interactions, appearance, or catabolism of the holo-phycobiliprotein fusion protein, particularly within the cell.
In another embodiment, the invention provides holo-phycobiliprotein based transcription reporter cells and assays. For example, the invention includes a recombinant cell which conditionally expresses a heterologous-to-the-cell, fluorescent, first holo-phycobiliprotein domain, wherein the cell makes and comprises components: a bilin, a recombinant bilin reductase, an apo-phycobiliprotein domain, and a recombinant phycobiliprotein domain-bilin lyase, wherein at least one of the reductase, apo-phycobiliprotein domain and lyase is expressed upon activation of a targeted transcriptional promoter, whereupon the components react inside the cell to form the holo-phycobiliprotein domain, which provides a reporter for the activation of the promoter.
REFERENCES:
patent: 2001/0055783 (2001-12-01), Allnutt et al.
Fairchild et al., PNAS, vol. 89, pp. 7017-7021, 1992.*
Frankenberg et al., The Plant Cell, vol. 13, pp. 965-978, Apr. 2001.*
Tooley et al., PNAS, vol. 98, No. 19, pp. 10560-10565, Sep. 2001.*
Landgraf et al., FEBS Letters, vol. 508, pp. 459-462, 2001.*
Allnutt et al. US 2001/055783, Dec. 27, 2001.
Gambetta G.A. et al. PNAS USA 98(19):10566-71, Sep. 11, 2001.
Frankenberg N. et al. Plant Cell 13(4):965-78, Apr. 2001.
Kronick M.N. et al. J. Immunol. Methods 92:1-13, Aug. 1986.
Fairchild C.D. et al. J. Biol. Chem. 269(12):8686-94, Mar. 25, 1994.
Cai Yuping
Glazer Alexander N.
Tooley Aaron J.
Carlson Karen Cochrane
Osman Richard Aron
Robinson Hope A.
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