Serca2a-Phospholamban Interaction Monitored by an Interposed Circularly Permutated Green Fluorescent Protein

2021 ◽  
Author(s):  
Maren E Arnold ◽  
Wolfgang R Dostmann ◽  
Jody Martin ◽  
Michael J Previs ◽  
Bradley Palmer ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Recombinant Protein ◽  
Integrated Assessment ◽  
Fluorescent Protein ◽  
Cardiac Contractility ◽  
Fluorescence Yield ◽  
Selective Interference ◽  
General Activation ◽  
Green Fluorescent ◽  
And Function

Background: The interaction of phospholamban (PLB) and the sarcoplasmic reticulum Ca2+-ATPase (Serca2a) is a key regulator of cardiac contractility and a therapeutic target in heart failure (HF). PLB mediated increases in Serca2a activity improve cardiac function and HF. Clinically this mechanism can only be exploited by a general activation of the proteinkinase A (PKA) which is associated with side effects and adverse clinical outcomes. A selective interference of the PLB-Serca2a interaction is desirable but will require novel tools that allow for an integrated assessment of this interaction under both physiological and pathophysiological conditions. Methods: A circularly permutated green fluorescent protein (cpGFP) was interposed between Serca2a and PLB to result into a single Serca2a-cpGFP-PLB recombinant protein (SGP). Expression, phosphorylation, fluorescence and function of SGP were evaluated. Results: Expression of SGP-cDNA results in a functional recombinant protein at the predicted molecular weight. The PLB domain of SGP retains its ability to polymerize and can be phosphorylated by PKA activation. This increases the fluorescent yield of SGP by between 10% to 165% depending on cell line and conditions. Summary: A single recombinant fusion protein that combines Serca2a, a circularly permutated green fluorescent protein and PLB can be expressed in cells and can be phosphorylated at the PLB domain which markedly increases the fluorescence yield. SGP is a novel cellular Serca2a-PLB interaction monitor.

scholarly journals Effects of Recombinant Protein Expression on Green Fluorescent Protein Diffusion inEscherichia coli

Biochemistry ◽  
2009 ◽  
Vol 48 (23) ◽  
pp. 5083-5089 ◽  
Author(s):  
Kristin M. Slade ◽  
Rachael Baker ◽  
Michael Chua ◽  
Nancy L. Thompson ◽  
Gary J. Pielak
Keyword(s):  
Green Fluorescent Protein ◽  
Recombinant Protein ◽  
Protein Expression ◽  
Fluorescent Protein ◽  
Recombinant Protein Expression ◽  
Inescherichia Coli ◽  
Protein Diffusion ◽  
Green Fluorescent

scholarly journals Transgenic Mice Expressing Green Fluorescent Protein under the Control of the Corticotropin-Releasing Hormone Promoter

Endocrinology ◽  
2009 ◽  
Vol 150 (12) ◽  
pp. 5626-5632 ◽  
Author(s):  
Tamar Alon ◽  
Ligang Zhou ◽  
Cristian A. Pérez ◽  
Alastair S. Garfield ◽  
Jeffrey M. Friedman ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Artificial Chromosome ◽  
Brain Regions ◽  
Ingestive Behavior ◽  
Physiological Processes ◽  
Functional Relevance ◽  
Green Fluorescent ◽  
And Function ◽  
The Brain

Abstract CRH is widely expressed in the brain and is of broad functional relevance to a number of physiological processes, including stress response, parturition, immune response, and ingestive behavior. To delineate further the organization of the central CRH network, we generated mice expressing green fluorescent protein (GFP) under the control of the CRH promoter, using bacterial artificial chromosome technology. Here we validate CRH-GFP transgene expression within specific brain regions and confirm the distribution of central GFP-producing cells to faithfully recapitulate that of CRH-expressing cells. Furthermore, we confirm the functional integrity of a population of GFP-producing cells by demonstrating their apposite responsiveness to nutritional status. We anticipate that this transgenic model will lend itself as a highly tractable tool for the investigation of CRH expression and function in discrete brain regions.

Green-fluorescent protein from the bioluminescent jellyfish Clytia gregaria: cDNA cloning, expression, and characterization of novel recombinant protein

2010 ◽  
Vol 9 (6) ◽  
pp. 757 ◽  
Author(s):  
Svetlana V. Markova ◽  
Ludmila P. Burakova ◽  
Ludmila A. Frank ◽  
Stefan Golz ◽  
Kseniya A. Korostileva ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Recombinant Protein ◽  
Cdna Cloning ◽  
Fluorescent Protein ◽  
Green Fluorescent

scholarly journals Construction and function of a recombinant adenovirus encoding a human aquaporin 1-green fluorescent protein fusion product

2000 ◽  
Vol 7 (3) ◽  
pp. 476-485 ◽  
Author(s):  
A T M Shamsul Hoque ◽  
Xibao Liu ◽  
Hideaki Kagami ◽  
William D Swaim ◽  
Robert B Wellner ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Recombinant Adenovirus ◽  
Fusion Product ◽  
Green Fluorescent Protein Fusion ◽  
Protein Fusion ◽  
Aquaporin 1 ◽  
Green Fluorescent ◽  
And Function

scholarly journals Construction of model strain of yeast Saccharomyces cerevisiae with regulated expression of recombinant human alpha-synuclein

2021 ◽  
Vol 15 (3) ◽  
pp. 41-50
Author(s):  
N. V. Hrushanyk ◽  
◽  
Y. I. Fedorko ◽  
O. V. Stasyk ◽  
O. G. Stasyk ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Green Fluorescent Protein ◽  
Recombinant Protein ◽  
Recombinant Strain ◽  
Lipid Membrane ◽  
Fluorescent Protein ◽  
Intracellular Localization ◽  
Alpha Synuclein ◽  
Yeast Cells ◽  
Green Fluorescent

Background. Improper folding and accumulation of a-synuclein aggregates are among the causes of Parkinson’s disease. The most important factor influencing the process of α-synuclein aggregation is the level of this protein in neurons which depends on the balance between its synthesis, degradation and secretion. Under certain conditions, when α-synuclein is synthesized at a high level, monomers of this protein can aggregate on the lipid membrane, which leads to the formation of amyloids, fibrils and protofibrils unable to perform their physiological functions. Since it is virtually impossible to study the properties of α-synuclein in vivo, researchers are actively using model biological systems (single-celled microorganisms, human cell lines, animal models etc.). The aim of this study was to construct a recombinant strain of Saccharomyces cerevisiae with controlled expression of human α-synuclein to study the regulation and properties of this protein and for screening for new low molecular weight chemi­cal compounds which can induce α-synuclein aggregation and/or degradation. Materials and methods. A recombinant strain of S. cerevisiae with controlled expression of α-synuclein conjugated to a green fluorescent protein was isolated. Western blotting with specific anti-α-synuclein antibodies was used to detect recombinant α-synuclein in yeast cells. Intracellular localization of heterologous chimeric green fluorescent protein conjugated to α-synuclein was also examined by fluorescence microscopy. Results. To construct a recombinant strain of S. cerevisiae, the coding sequence of the human wild-type α-synuclein gene was expressed under the regulated promoter of the ScMET25 gene. Analysis of the effect of different concentrations of exogenous methionine as a factor regulating the expression of the ScMET25 promoter on the content of recombinant protein showed that the expression of the human α-synuclein gene in S. cerevisiae is repressed in the presence of methionine at a concentration of 10 mg/L and higher. During long-term cultivation of yeast cells, this effect decreased due to the depletion of methionine in the growth medium. As a result, recombinant protein synthesis was restored, and α-synuclein content in such cells approached that of cells grown in a medium with a low concentration of (5 mg/L), or without methionine. It was also found that overproduction of recombinant α-synuclein in S. cerevisiae cells had virtually no effect on culture growth, indicating the absence or a very weak toxic effect of human α-synuclein on yeast physiology. Conclusions. The obtained data indicate a concentration-dependent effect of methionine on the level of recombinant α-synuclein synthesis in S. cerevisiae yeast cells. Such controlled expression of the studied protein can be used to screen for compounds capable of promoting dose-dependent aggregation or degradation of α-synuclein in yeast cells and potentially in human cells as well.

scholarly journals The jellyfish green fluorescent protein: A new tool for studying ion channel expression and function

Neuron ◽  
1995 ◽  
Vol 14 (2) ◽  
pp. 211-215 ◽  
Author(s):  
John Marshall ◽  
Raymond Molloy ◽  
Guy W.J Moss ◽  
James R Howe ◽  
Thomas E Hughes
Keyword(s):  
Green Fluorescent Protein ◽  
Ion Channel ◽  
Fluorescent Protein ◽  
Protein A ◽  
Channel Expression ◽  
Green Fluorescent ◽  
And Function

scholarly journals Astrocyte-to-neuron transportation of Enhanced Green Fluorescent Protein in cerebral cortex requires F-actin dependent Tunneling Nanotubes

2021 ◽  
Author(s):  
Jing Chen ◽  
Junyan Cao
Keyword(s):  
Cerebral Cortex ◽  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Cell Contact ◽  
Neural Cells ◽  
Tunneling Nanotubes ◽  
Green Fluorescent ◽  
And Function

Abstract Tunneling Nanotube (TNT) , a dynamic cell-cell contact, is dependent on actin polimerization. TNTs are efficient in transporting ions, proteins and organelles intercellularly, which are important mechanisms in physiological and phathological precesses. Reported studies on the existence and function of TNTs among neural cells focus on cultured cell for the convenience in detecting TNTs’ ultrastructure. In this study, the adeno-associated virus (AAV-GFAP-EGFP-p2A-cre) was injected in the cerebral cortex of knock-in mice ROSA26 GNZ. GFAP promoter initiated the expression of Enhanced Green Fluorescent Protein (EGFP) in infected astrocytes. At 10 days post injection (10 DPI), we found that EGFP could transfer from astrocytes in layerⅠ-Ⅲ to neurons in layer Ⅴ. The dissemination of EGFP was not through endocytosis or exosome. And the intercellular transportation of EGFP was F-actin dependent. Therefore, we concluded EGFP transported from astrocytes to neurons in coetex via F-actin dependent TNTs. Although it is hardly to detect the ultrastrucute of TNTs in brain for its transiency and the noisy background, we established an animal model and indirect experimental methods to explore TNTs in vivo.

scholarly journals A Rewired Green Fluorescent Protein: Folding and Function in a Nonsequential, Noncircular GFP Permutant

Biochemistry ◽  
2010 ◽  
Vol 49 (51) ◽  
pp. 10773-10779 ◽  
Author(s):  
Philippa J. Reeder ◽  
Yao-Ming Huang ◽  
Jonathan S. Dordick ◽  
Christopher Bystroff
Keyword(s):  
Protein Folding ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Green Fluorescent ◽  
And Function
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