ArticleProtein expression and purification2007
Profiling of membrane protein variants in a baculovirus system by coupling cell-surface detection with small-scale parallel expression.
Article in Protein expression and purification, 2007. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 18 papers.
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Who cites it
18 citing papers in PubMed, 39 citations in OpenAlex.
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- Overexpression and purification of Dicer and accessory proteins for biochemical and structural studies.Methods (San Diego, Calif.) · 2017Article
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- Mechanism of B-box 2 domain-mediated higher-order assembly of the retroviral restriction factor TRIM5α.eLife · 2016Article
- In vitro expression and analysis of the 826 human G protein-coupled receptors.Protein & cell · 2016Article
- Large-scale production and protein engineering of G protein-coupled receptors for structural studies.Frontiers in pharmacology · 2015Review
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- Ice breaking in GPCR structural biology.Acta pharmacologica Sinica · 2012Review
- GPCR stabilization using the bicelle-like architecture of mixed sterol-detergent micelles.Methods (San Diego, Calif.) · 2011Article
- High throughput platforms for structural genomics of integral membrane proteins.Current opinion in structural biology · 2011Review
- Evaluation of the Pichia pastoris expression system for the production of GPCRs for structural analysis.Microbial cell factories · 2011Article
- Unlocking the eukaryotic membrane protein structural proteome.Current opinion in structural biology · 2010Review
- Ligand binding and subtype selectivity of the human A(2A) adenosine receptor: identification and characterization of essential amino acid residues.The Journal of biological chemistry · 2010Article
- Stabilization of the human beta2-adrenergic receptor TM4-TM3-TM5 helix interface by mutagenesis of Glu122(3.41), a critical residue in GPCR structure.Journal of molecular biology · 2008Article
Corrections and comments
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Authors and funding
17 authors at 2 institutions in 1 country.
Funding
Abstract
Production of structure-grade mammalian membrane proteins in substantial quantities has been hindered by a lack of methods for effectively profiling multiple constructs expression in higher eukaryotic systems such as insect or mammalian cells. To address this problem, a specialized small-scale eukaryotic expression platform by Thomson Instrument Company (Vertiga-IM) was developed and used in tandem with a Guava EasyCyte microcapillary 96-well cytometer to monitor cell density and health and evaluate membrane protein expression. Two proof of concept experiments were conducted using the human beta(2)-adrenergic receptor (beta(2)AR) and the gap junction protein connexin26 (Cx26) in a baculovirus expression system. First, cell surface expression was used to assess the expression levels of 14 beta(2)AR truncation variants expressed using the Vertiga-IM shaker. Three of these variants were then compared to wild-type beta(2)AR using three metrics: cell surface expression, saturation ligand binding and protein immunoblot analysis of dodecylmaltoside extracted material. Second, a series of systematic Cx26 truncation variants were evaluated for expression by protein immunoblot analysis. The cumulative results for these two systems show that the Vertiga-IM instrument can be used effectively in the parallel insect cell microexpression of membrane protein variants, and that the expression of cell surface molecules as monitored with the Guava EasyCyte instrument can be used to rapidly assess the production of properly folded proteins in the baculovirus expression system. This approach expedites the in vitro evaluation of a large number of mammalian membrane protein variants.
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