Functional expression and direct visualization of the human α2B-adrenergic receptor and α2B-AR-green fluorescent fusion protein in mammalian cell using Semliki Forest virus vectors

Sen, Satyabrata; Jaakola, Veli-Pekka; Heimo, Heikki; Engström, Mia; Larjomaa, Pauliina; Scheinin, Mika; Lundström, Kenneth; Goldman, Adrian

doi:10.1016/j.pep.2003.08.006

Cited by 20 publications

(10 citation statements)

References 35 publications

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“…Production through cell-free expression systems has proven useful for structural determinations of both soluble and membrane protein targets (Chen et al, 2007b), but large-scale use of these techniques for GPCR expression have yet to yield a GPCR structure (Klammt et al, 2007a,b;Junge et al, 2008Junge et al, , 2010. A variety of mammalian cell-based protocols using transfected and virally infected cells to express GPCRs for biochemical as well as structural determination studies have been published (Sen et al, 2003;Shukla et al, 2006a,b), but rhodopsin mutants expressed in transfected COS-1 cells constitute the only success for this expression methodology (Standfuss et al, 2007(Standfuss et al, , 2011Stenkamp, 2008). The most efficient and successful method to date, apart from native expression, has been the expression of truncated/stabilized/engineered GPCR constructs in baculovirus-infected insect cell hosts Rosenbaum et al, 2007;Roth et al, 2008;Warne et al, 2008Warne et al, , 2009Tate and Schertler, 2009).…”

Section: A G Protein-coupled Receptor Construct Design and Expressionmentioning

confidence: 99%

The Significance of G Protein-Coupled Receptor Crystallography for Drug Discovery

2011

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Section: A G Protein-coupled Receptor Construct Design and Expressionmentioning

confidence: 99%

The Significance of G Protein-Coupled Receptor Crystallography for Drug Discovery

2011

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“…Indeed, where experiments have been performed to look at the functionality of the expressed membrane protein, often only a small percentage of the protein is functional. For example, high levels of intracellular retention were observed for SFV-expressed α 2 adrenergic receptor (Sen et al 2003), the bradykinin B 2 receptor (Shukla et al 2006a) and the angiotensin II receptor (Shukla et al 2006b). Only 0.5% and 7% of the ion channels P2X 2 and HCN2, respectively, were located in the plasma membrane after expression using SFV (Eifler et al 2007).…”

Section: Choice Of Mammalian Expression Systemmentioning

confidence: 99%

Overexpression of membrane proteins in mammalian cells for structural studies

Andréll

Tate

2012

Molecular Membrane Biology

107

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The number of structures of integral membrane proteins from higher eukaryotes is steadily increasing due to a number of innovative protein engineering and crystallization strategies devised over the last few years. However, it is sobering to reflect that these structures represent only a tiny proportion of the total number of membrane proteins encoded by a mammalian genome. In addition, the structures determined to date are of the most tractable membrane proteins, i.e., those that are expressed functionally and to high levels in yeast or in insect cells using the baculovirus expression system. However, some membrane proteins that are expressed inefficiently in these systems can be produced at sufficiently high levels in mammalian cells to allow structure determination. Mammalian expression systems are an under-used resource in structural biology and represent an effective way to produce fully functional membrane proteins for structural studies. This review will discuss examples of vertebrate membrane protein overexpression in mammalian cells using a variety of viral, constitutive or inducible expression systems.

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“…In this context, especially SFV vectors have been used for the expression of topologically different proteins [14,19]. Particularly, the expression of membrane proteins has been very successful indicated by the expression of G protein-coupled receptors both at high levels [20] and in large numbers [21]. Moreover, they have demonstrated neuronspecific gene expression in primary neurons [22], in hippocampal slice cultures [23] and in vivo in rodent brain [24].…”

Section: Basics Of Alphavirusesmentioning

confidence: 99%

Alphavirus Vectors for Therapy of Neurological Disorders

Lundström¹

2012

J Stem Cell Res Ther

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Alphavirus vectors engineered for gene delivery and expression of heterologous proteins have been considered as valuable tools for research on neurological disorders. They possess a highly efficient susceptibility for neuronal cells and can provide extreme levels of heterologous gene expression. However, they generally generate short-term transient expression, which might limit their therapeutic use in many neurological disorders often requiring long-term even life-long presence of therapeutic agents. Recent development in gene silencing applying both RNA interference and microRNA approaches will certainly expand the application range. Moreover, alphaviruses provide interesting models for neurological diseases such as demyelinating and spinal motor diseases.

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Functional expression and direct visualization of the human α2B-adrenergic receptor and α2B-AR-green fluorescent fusion protein in mammalian cell using Semliki Forest virus vectors

Cited by 20 publications

References 35 publications

The Significance of G Protein-Coupled Receptor Crystallography for Drug Discovery

The Significance of G Protein-Coupled Receptor Crystallography for Drug Discovery

Overexpression of membrane proteins in mammalian cells for structural studies

Alphavirus Vectors for Therapy of Neurological Disorders

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