Rapid protein-folding assay using green fluorescent protein

Waldo, Geoffrey S.; Standish, Blake M.; Berendzen, Joel; Terwilliger, Thomas C.

doi:10.1038/10904

Cited by 834 publications

(783 citation statements)

References 28 publications

Supporting

Mentioning

754

Contrasting

Unclassified

Order By: Relevance

“…28,29 Our results suggest that the quenching peptide is not well-folded, which would therefore lead to a less stably folded intermediate of the GFP barrel in nCA-GFP.…”

Section: Discussionmentioning

confidence: 96%

Structural basis of fluorescence quenching in caspase activatable‐GFP

Nicholls

Hardy

2013

Protein Science

View full text Add to dashboard Cite

Apoptosis is critical for organismal homeostasis and a wide variety of diseases. Caspases are the ultimate executors of the apoptotic programmed cell death pathway. As caspases play such a central role in apoptosis, there is significant demand for technologies to monitor caspase function. We recently developed a caspase activatable-GFP (CA-GFP) reporter. CA-GFP is unique due to its ''dark'' state, where chromophore maturation of the GFP is inhibited by the presence of a C-terminal peptide. Here we show that chromophore maturation is prevented because CA-GFP does not fold into the robust b-barrel of GFP until the peptide has been cleaved by active caspase. Both CA-GFP and GFP 1-10 , a split form of GFP lacking the 11th strand, have similar secondary structure, different from mature GFP. A similar susceptibility to proteolytic digestion indicates that this shared structure is not the robust, fully formed GFP b-barrel. We have developed a model that suggests that as CA-GFP is translated in vivo it follows the same folding path as wild-type GFP; however, the presence of the appended peptide does not allow CA-GFP to form the barrel of the fully matured GFP. CA-GFP is therefore held in a ''pro-folding'' intermediate state until the peptide is released, allowing it to continue folding into the mature barrel geometry. This new understanding of the structural basis of the dark state of the CA-GFP reporter will enable manipulation of this mechanism in the development of reporter systems for any number of cellular processes involving proteases and potentially other enzymes.

show abstract

“…28,29 Our results suggest that the quenching peptide is not well-folded, which would therefore lead to a less stably folded intermediate of the GFP barrel in nCA-GFP.…”

Section: Discussionmentioning

confidence: 96%

Structural basis of fluorescence quenching in caspase activatable‐GFP

Nicholls

Hardy

2013

Protein Science

View full text Add to dashboard Cite

show abstract

“…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%

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

Bleckmann

Schmelz

Schinkowski

et al. 2016

Biotech & Bioengineering

View full text Add to dashboard Cite

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.

show abstract

“…All fluorescence data were processed using a method adapted from Waldo et al 34 To account for handling variability in cell resuspension, the fluorescent values were normalized against the OD 600 measurement F N ¼ F=OD 600 Þ. Furthermore, to account for the inherent fluorescence of E. coli, all fluorescent values are reported as a relative fluorescence RF t ¼ F Nt =F N0 , where the normalized fluorescence at a given time point is divided by the normalized fluorescence of noninduced cells harboring the same plasmid.…”

Section: Methodsmentioning

confidence: 99%

Bridging the gap: A GFP‐based strategy for overexpression and purification of membrane proteins with intra and extracellular C‐termini

et al. 2010

View full text Add to dashboard Cite

Low expression and instability during isolation are major obstacles preventing adequate structure-function characterization of membrane proteins (MPs). To increase the likelihood of generating large quantities of protein, C-terminally fused green fluorescent protein (GFP) is commonly used as a reporter for monitoring expression and evaluating purification. This technique has mainly been restricted to MPs with intracellular C-termini (C in ) due to GFP's inability to fluoresce in the Escherichia coli periplasm. With the aid of Glycophorin A, a single transmembrane spanning protein, we developed a method to convert MPs with extracellular C-termini (C out ) to C in ones providing a conduit for implementing GFP reporting. We tested this method on eleven MPs with predicted C out topology resulting in high level expression. For nine of the eleven MPs, a stable, monodisperse protein-detergent complex was identified using an extended fluorescencedetection size exclusion chromatography procedure that monitors protein stability over time, a critical parameter affecting the success of structure-function studies. Five MPs were successfully cleaved from the GFP tag by site-specific proteolysis and purified to homogeneity. To address the challenge of inefficient proteolysis, we explored expression and purification conditions in the absence of the fusion tag. Contrary to previous studies, optimal expression conditions established with the fusion were not directly transferable for overexpression in the absence of the GFP tag. These studies establish a broadly applicable method for GFP screening of MPs with C out topology, yielding sufficient protein suitable for structure-function studies and are superior to expression and purification in the absence GFP fusion tagging.

show abstract

Rapid protein-folding assay using green fluorescent protein

Cited by 834 publications

References 28 publications

Structural basis of fluorescence quenching in caspase activatable‐GFP

Structural basis of fluorescence quenching in caspase activatable‐GFP

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

Bridging the gap: A GFP‐based strategy for overexpression and purification of membrane proteins with intra and extracellular C‐termini

Contact Info

Product

Resources

About