Menu

Detecting Protein Clumps

A synthetic genetic tool called yTRAP allows high-throughput detection of protein aggregates in cells.  

Feb 1, 2018
Ruth Williams

AGGREGATION ASSAY: To detect clumping of a protein of interest, express it together with a synthetic transcriptional activator domain (grey). If the protein remains soluble, the reporter gene (green), which is under the control of a synthetic promoter that corresponds to the activator, will be expressed. If the proteins clump together, it will not. © GEORGE RETSECK
See full infographic: WEB

The aggregation of cellular proteins into insoluble clumps is a hallmark of many diseases, including Alzheimer’s, Parkinson’s, systemic amyloidosis, prion diseases, and type 2 diabetes. Protein agglomeration can also be a feature of normal cellular functions, such as signal transduction, synapse modification, and the regulation of RNAs during cellular stress.

Tools for studying such physiological and pathological protein aggregations, however, are limited, explains biomedical engineer Ahmad Khalil of Boston University. The principal options for researchers, he says, are either to destroy cells and analyze their innards for protein aggregates, or append a fluorescent tag to the proteins of interest within cells and view the formation of clumps (bright spots) with a microscope.

While this second option maintains the protein’s normal physiological surroundings, Khalil says, “inherently it is not a very high-throughput way of studying this phenomenon.”

Khalil and colleagues’ new approach, called yeast transcriptional reporting of aggregating proteins (yTRAP), allows for high-throughput analysis and doesn’t destroy cells. An aggregation-prone protein of interest is first fused to a synthetic transcriptional activator, which can drive gene expression from a synthetic promoter only when the protein is not aggregated. Linking the synthetic promoter to a fluorescent reporter allows easy identification and, if desired, sorting of cells with and without aggregates.

The team has used yTRAP to detect accumulations of prions and other proteins in yeast; to perform a high-throughput screen for aggregation-preventing mutants; and to identify cells that “remembered” an environmental stimulus (heat)—using a yeast strain engineered to express a stably aggregating prion under the control of a heat-responsive promoter.

The assay “can inform us about a very important cellular process,” says Madan Babu of the MRC Laboratory of Molecular Biology in Cambridge, U.K., who was not involved with the project. But it also “can be applied to a number of different questions,” he says. “It’s a bit of a tour de force.” (Cell, 171:966–79, 2017) 

AGGREGATION DETECTION TECHNIQUE HOW IT WORKS ASSAY EQUIPMENT HIGH THROUGHPUT CAPACITY CELL TYPES
Fluorescent tagging of aggregation-prone protein
 
Proteins are fluorescently tagged—either by being engineered as fluorescent fusion proteins, or by applying fluorescent antibodies. Soluble proteins appear as diffuse fluorescence, while aggregates appear as bright spots.
 
Fluorescence
microscope
 
Poor Any
yTRAP Proteins are fused to a synthetic transcriptional activator. The fusion protein is then expressed in cells where corresponding synthetic promoters drive expression of reporters. Soluble proteins activate the reporter, while aggregated proteins do not. Fluorescence microscope, flow cytometer, fluorescence-
activated cell sorter (FACS), or fluorescence microplate reader
Good Limited to yeast so far, but future versions are planned
for other cell types

 

September 2018

The Muscle Issue

The dynamic tissue reveals its secrets

Marketplace

Sponsored Product Updates

StemExpress LeukopakâNow Available in Frozen Format

StemExpress LeukopakâNow Available in Frozen Format

StemExpress, a Folsom, California based leading supplier of human biospecimens, announces the release of frozen Peripheral Blood Leukopaks. Leukopaks provide an enriched source of peripheral blood mononuclear cells (PBMCs) with low granulocyte and red blood cells that can be used in a variety of downstream cell-based applications.

New Antifade Mounting Media from Vector Laboratories Enhances Immunofluorescence Applications

New Antifade Mounting Media from Vector Laboratories Enhances Immunofluorescence Applications

Vector Laboratories, a leader in the development and manufacture of labeling and detection reagents for biomedical research, introduces VECTASHIELD® Vibrance™ – antifade mounting media that delivers significant improvements to the immunofluorescence workflow.

Best Practices for Sample Preparation and Lipid Extraction from Various Samples

Best Practices for Sample Preparation and Lipid Extraction from Various Samples

Download this white paper from Bertin Technologies to learn how to extract and analyze lipid samples from various models!

Bio-Rad Launches CHT Ceramic Hydroxyapatite XT Media and Nuvia HP-Q Resin for Process Protein Purification

Bio-Rad Launches CHT Ceramic Hydroxyapatite XT Media and Nuvia HP-Q Resin for Process Protein Purification

Bio-Rad Laboratories, Inc. (NYSE: BIO and BIOb), a global leader of life science research and clinical diagnostic products, today announced the launch of two new chromatography media for process protein purification: CHT Ceramic Hydroxyapatite XT Media and Nuvia HP-Q Resin.