Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein

Cabantous, Stéphanie; Terwilliger, Thomas C.; Waldo, Geoffrey S.

doi:10.1038/nbt1044

Cited by 827 publications

(1,025 citation statements)

References 38 publications

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“…Because of the paucity of data concerning the topology of chloroplast Omp85 proteins and the importance of the POTRA domain as a central functional element in the models of chloroplast OMP integration/assembly and preprotein import, we decided to investigate the topology of the atToc75-III (AT3G46740) and atToc75-V (AT5G19620). We applied the recently established self-assembling split GFP (saGFP) (34), which has been used to determine membrane protein topology in vivo before (35). Here, the 11-stranded β-barrel of GFP was split into saGFP1-10, comprising β-strands 1-10 (short 1-10), and a saGFP11 (short 11) fragment, containing β-strand 11.…”

mentioning

confidence: 99%

Chloroplast Omp85 proteins change orientation during evolution

Sommer

Daum

Groß

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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The majority of outer membrane proteins (OMPs) from Gramnegative bacteria and many of mitochondria and chloroplasts are β-barrels. Insertion and assembly of these proteins are catalyzed by the Omp85 protein family in a seemingly conserved process. All members of this family exhibit a characteristic N-terminal polypeptide-transport-associated (POTRA) and a C-terminal 16-stranded β-barrel domain. In plants, two phylogenetically distinct and essential Omp85's exist in the chloroplast outer membrane, namely Toc75-III and Toc75-V. Whereas Toc75-V, similar to the mitochondrial Sam50, is thought to possess the original bacterial function, its homolog, Toc75-III, evolved to the pore-forming unit of the TOC translocon for preprotein import. In all current models of OMP biogenesis and preprotein translocation, a topology of Omp85 with the POTRA domain in the periplasm or intermembrane space is assumed. Using selfassembly GFP-based in vivo experiments and in situ topology studies by electron cryotomography, we show that the POTRA domains of both Toc75-III and Toc75-V are exposed to the cytoplasm. This unexpected finding explains many experimental observations and requires a reevaluation of current models of OMP biogenesis and TOC complex function.

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mentioning

confidence: 99%

Chloroplast Omp85 proteins change orientation during evolution

Sommer

Daum

Groß

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…The DNA sequences of spl GFP1‐10 and spl GFP11 were designed according to Waldo et al (1999) (Cabantous et al, 2005b) and synthesized by Genscript (Piscataway, NJ). The synthesized constructs as well as the vector OpIE2‐eGFP (Bleckmann et al, 2015) were digested with BamH I and Avr II and ligated following a standard protocol resulting in the OpIE2‐ spl GFP1‐10 vector.…”

Section: Methodsmentioning

confidence: 99%

“…Transient co‐transfection in insect cells using the SplitGFP system developed by Cabantous and coworkers (Cabantous et al, 2005b) for E. coli expression screens meets these demands. Merely one β ‐strand of a specially developed GFP (Cabantous and Waldo, 2006) ( spl GFP11) is fused to the target protein and is able to reassemble with the co‐expressed residual part of GFP ( spl GFP1‐10) of the fluorescence‐competent spl GFP molecule.…”

Section: Introductionmentioning

confidence: 99%

“…Merely one β ‐strand of a specially developed GFP (Cabantous and Waldo, 2006) ( spl GFP11) is fused to the target protein and is able to reassemble with the co‐expressed residual part of GFP ( spl GFP1‐10) of the fluorescence‐competent spl GFP molecule. The assembly of the spl GFP11 β‐strand is essential to trigger the folding step for generation of the cyclized chromophore of spl GFP (Cabantous et al, 2005b). …”

Section: Introductionmentioning

confidence: 99%

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Fast plasmid based protein expression analysis in insect cells using an automated SplitGFP screen

Bleckmann

Schmelz

Schinkowski

et al. 2016

Biotech & Bioengineering

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Recombinant protein expression often presents a bottleneck for the production of proteins for use in many areas of animal‐cell biotechnology. Difficult‐to‐express proteins require the generation of numerous expression constructs, where popular prokaryotic screening systems often fail to identify expression of multi domain or full‐length protein constructs. Post‐translational modified mammalian proteins require an alternative host system such as insect cells using the Baculovirus Expression Vector System (BEVS). Unfortunately this is time‐, labor‐, and cost‐intensive. It is clearly desirable to find an automated and miniaturized fast multi‐sample screening method for protein expression in such systems. With this in mind, in this paper a high‐throughput initial expression screening method is described using an automated Microcultivation system in conjunction with fast plasmid based transient transfection in insect cells for the efficient generation of protein constructs. The applicability of the system is demonstrated for the difficult to express Nucleotide‐binding Oligomerization Domain‐containing protein 2 (NOD2). To enable detection of proper protein expression the rather weak plasmid based expression has been improved by a sensitive inline detection system. Here we present the functionality and application of the sensitive SplitGFP (split green fluorescent protein) detection system in insect cells. The successful expression of constructs is monitored by direct measurement of the fluorescence in the BioLector Microcultivation system. Additionally, we show that the results obtained with our plasmid‐based SplitGFP protein expression screen correlate directly to the level of soluble protein produced in BEVS. In conclusion our automated SplitGFP screen outlines a sensitive, fast and reliable method reducing the time and costs required for identifying the optimal expression construct prior to large scale protein production in baculovirus infected insect cells. Biotechnol. Bioeng. 2016;113: 1975–1983. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

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“…Other methods exploiting the ability of GFP moieties to be recombined to form a FP in living cells (bimolecular complementation or split-GFP method) [91] can also be used to monitor ERAD. In this technique, the C-terminal β-strand of GFP (S11) is fused to an ERAD substrate such as CD3δ.…”

Section: Approaches For Imaging Er Stress and Upr Activity In Livinmentioning

confidence: 99%

Approaches to imaging unfolded secretory protein stress in living cells

Lajoie

Fazio

Snapp

2014

Endoplasmic Reticulum Stress in Diseases

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The endoplasmic reticulum (ER) is the point of entry of proteins into the secretory pathway. Nascent peptides interact with the ER quality control machinery that ensures correct folding of the nascent proteins. Failure to properly fold proteins can lead to loss of protein function and cytotoxic aggregation of misfolded proteins that can lead to cell death. To cope with increases in the ER unfolded secretory protein burden, cells have evolved the Unfolded Protein Response (UPR). The UPR is the primary signaling pathway that monitors the state of the ER folding environment. When the unfolded protein burden overwhelms the capacity of the ER quality control machinery, a state termed ER stress, sensor proteins detect accumulation of misfolded peptides and trigger the UPR transcriptional response. The UPR, which is conserved from yeast to mammals, consists of an ensemble of complex signaling pathways that aims at adapting the ER to the new misfolded protein load. To determine how different factors impact the ER folding environment, various tools and assays have been developed. In this review, we discuss recent advances in live cell imaging reporters and model systems that enable researchers to monitor changes in the unfolded secretory protein burden and activation of the UPR and its associated signaling pathways.

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Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein

Cited by 827 publications

References 38 publications

Chloroplast Omp85 proteins change orientation during evolution

Chloroplast Omp85 proteins change orientation during evolution

Fast plasmid based protein expression analysis in insect cells using an automated SplitGFP screen

Approaches to imaging unfolded secretory protein stress in living cells

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