Genetically Fused Protein A–Luciferase for Immunological Blotting Analyses

Zhang, Xiaomei; Kobatake, Eiry; Kobayashi, Kiyoaki; Yanagida, Yasuko; Aizawa, Masuo

doi:10.1006/abio.2000.4584

Cited by 19 publications

(11 citation statements)

References 14 publications

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“…5, detection of cell surface ATP was performed using proA-luc, a fusion protein between staphylococcal protein A and firefly luciferase (21). Protein A specifically binds to the Fc domain of immunoglobulin G (IgG) and, as mentioned before, firefly luciferase can be used to detect ATP by luminescence emission.…”

Section: Methodsmentioning

confidence: 99%

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Homeostasis of Extracellular ATP in Human Erythrocytes

Montalbetti

Denis

Pignataro

et al. 2011

Journal of Biological Chemistry

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We explored the intra-and extracellular processes governing the kinetics of extracellular ATP (ATPe) in human erythrocytes stimulated with agents that increase cAMP. Using the luciferinluciferase reaction in off-line luminometry we found both direct adenylyl cyclase activation by forskolin and indirect activation through ␤-adrenergic stimulation with isoproterenol-enhanced [ATP]e in a concentration-dependent manner. A mixture (3V) containing a combination of these agents and the phosphodiesterase inhibitor papaverine activated ATP release, leading to a 3-fold increase in [ATP]e, and caused increases in cAMP concentration (3-fold for forskolin ؉ papaverine, and 10-fold for 3V). The pannexin 1 inhibitor carbenoxolone and a pannexin 1 blocking peptide ( Both for soluble luciferase and proA-luc, ATP efflux was fully blocked by carbenoxolone, pointing to a 3V-induced mechanism of ATP release mediated by pannexin 1. Ecto-ATPase activity was extremely low (ϳ28 fmol ؋ (10 6 cells min) ؊1 ), but nevertheless physiologically relevant considering the high density of erythrocytes in human blood.All cell types appear to possess mechanisms that enable a controlled, nonlytic release of ATP, that is, a release not involving cell membrane rupture, and which occurs in response to osmotic, mechanical, and/or neurohormonal stimuli (1). Specifically human erythrocytes release ATP following exposure to ␤-adrenergic stimulation, mechanical deformation, reduced oxygen tension, or acidosis (2). All of these represent physiological conditions to which erythrocytes are exposed in the vasculature, e.g. when passing through constricted vessels or in the contracting striated muscle (3). Once in the extracellular medium, extracellular ATP (ATPe) 5 can trigger different cellular responses by interacting with P receptors on the cell surface while at the same time its concentration is controlled by the activities of one or more ectonucleotidases (4, 5). Several reports published over recent years have shown that an increase in intracellular cyclic AMP (cAMP) concentration triggered ATP release from human erythrocytes (6, 7). Receptor-mediated ATP release in human erythrocytes involves activation of heterotrimeric G proteins G s or G i/o (3, 8, 9). Regarding the G s pathway, activation of ␤-adrenergic receptors by various agonists was reported to stimulate adenylyl cyclase, with concomitant increases in cAMP levels and protein kinase A activity (6, 10). Moreover, direct activation of adenylyl cyclase by forskolin resulted in both ATP release and cAMP increases in human and rabbit erythrocytes (6).Despite the accumulated knowledge regarding the intracellular signaling events mediating ATP release, comparatively little is known regarding the processes governing the kinetics of ATPe accumulation at the surface in animal cells (11,12), with rates of intracellular ATP release and extracellular ATP hydrolysis being the main actors. The human non-nucleated erythrocyte is an excellent model in this respect, because it lacks intracellular compartments and...

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Section: Methodsmentioning

confidence: 99%

“…Expression and purification methods were described before (21,22,26). Briefly, the JM109 strain of Escherichia coli cells was transformed with pMALU7.…”

Section: Methodsmentioning

confidence: 99%

Homeostasis of Extracellular ATP in Human Erythrocytes

Montalbetti

Denis

Pignataro

et al. 2011

Journal of Biological Chemistry

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“…Recently, bioluminescence systems have been applied to detect other events, such as proteinprotein interactions and protease activity, by modification of luciferase. [6][7][8][9][10][11] On the other hand, functional luciferin analogues, the luminescence properties of which are changed dramatically by specific activity, for instance, b-galactosi-Abstract: Firefly luciferase is widely used as a reporter gene in assays to study gene expression, gene delivery, and so on because of its extremely high signal-to-noise ratio. The availability of a range of bioluminogenic substrates would greatly extend the applicability of the luciferin-luciferase system.…”

Section: Introductionmentioning

confidence: 99%

Aminoluciferins as Functional Bioluminogenic Substrates of Firefly Luciferase

Takakura

Kojima

Urano

et al. 2011

Chemistry An Asian Journal

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Firefly luciferase is widely used as a reporter gene in assays to study gene expression, gene delivery, and so on because of its extremely high signal-to-noise ratio. The availability of a range of bioluminogenic substrates would greatly extend the applicability of the luciferin-luciferase system. Herein, we describe a design concept for functional bioluminogenic substrates based on the aminoluciferin (AL) scaffold, together with a convenient, high-yield method for synthesizing N-alkylated ALs. We confirmed the usefulness of ALs as bioluminogenic substrates by synthesizing three probes. The first was a conjugate of AL with glutamate, Glu-AL. When Glu-AL, the first membrane-impermeable bioluminogenic substrate of luciferases, was applied to cells transfected with luciferase, luminescence was not observed; that is, by using Glu-AL, we can distinguish between intracellular and extracellular events. The second was Cy5-AL, which consisted of Cy5, a near-infrared (NIR) cyanine fluorescent dye, and AL, and emitted NIR light. When Cy5-AL reacted with luciferase, luminescence derived from Cy5 was observed as a result of bioluminescence resonance energy transfer (BRET) from AL to Cy5. The NIR emission wavelength would allow a signal to be observed from deeper tissues in bioluminescence in vivo imaging. The third was biotin-DEVD-AL (DEVD = the amino acid sequence Asp-Glu-Val-Asp), which employed a caspase-3 substrate peptide as a switch to control the accessibility of the substrate to luciferase, and could detect the activity of caspase-3 in a time-dependent manner. This generalized design strategy should be applicable to other proteases. Our results indicate that the AL scaffold is appropriate for a range of functional luminophores and represents a useful alternative substrate to luciferin.

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“…In addition, steric hindrance is often a problem in immunoassays; it affects the immunological reactions that occur due to random conjugation (Uhlén et al 1992;Zhang et al 2000). To obviate such problems, different strategies for the production of fusion proteins between SpA and enzymes for immunoassays have been reported (Frank et al 1996;Ståhl and Nygren et al 1997;Zhang et al 2000). Green fluorescent protein (GFP) has been used extensively in various fields of biological research.…”

Section: Introductionmentioning

confidence: 98%

Expression and secretion of recombinant ZZ–EGFP fusion protein by the methylotrophic yeast Pichia pastoris

et al. 2008

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We constructed a fusion protein ZZ-EGFP by fusing the ZZ domains of staphylococcal protein A (SpA) and enhanced green fluorescent protein (EGFP). ZZ-EGFP was secreted in the yeast, Pichia pastoris, with a hexahistidine tag. Its expression level was determined by measuring the fluorescence of EGFP. When the recombinant yeast cells in shake-flasks were induced with 0.5% methanol for 96 h, a maximum yield of 115 mg ZZ-EGFP/l was obtained. The resulting ZZ-EGFP fusion protein retained immunoglobulin G (IgG)-binding capacity and EGFP fluorescence. ZZ-EGFP was then used in immunofluorescence assays for detecting antinuclear antibodies (ANA); it produced a good signal that was comparable in its brightness and fluorescence pattern to that generated with fluorescein isothiocyanate (FITC)-labelled anti-human IgG. Thus, ZZ-EGFP showed great potential in immunological applications due to its ability to bind to various IgG from different animal sources.

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Genetically Fused Protein A–Luciferase for Immunological Blotting Analyses

Cited by 19 publications

References 14 publications

Homeostasis of Extracellular ATP in Human Erythrocytes

Homeostasis of Extracellular ATP in Human Erythrocytes

Aminoluciferins as Functional Bioluminogenic Substrates of Firefly Luciferase

Expression and secretion of recombinant ZZ–EGFP fusion protein by the methylotrophic yeast Pichia pastoris

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