CCD Imaging of BRET

Like FRET, BRET can be used to measure protein-protein interactions. Both resonance energy transfer techniques rely upon the proximity of donor and acceptor molecules. Whereas FRET involves fluorescent donor and acceptor molecules, BRET replaces the fluorescent donor molecule with a bioluminescent protein. When this donor protein is incubated with the appropriate substrate, the resulting chemiluminescence can excite the acceptor fluorophore, causing it to fluoresce at its characteristic emission wavelength. Like other bioluminescence-based strategies, BRET does not rely on excitation by an external light source. This characteristic can lead to a higher signal-to-background ratio than otherwise possible and is a reason why bioluminescence is a popular strategy for the low signal application of live animal imaging. Indeed, protein-protein interactions have been successfully imaged in small animals by a bioluminescence-based enzyme complementation (split luciferase) strategy. However, complementation suffers from low efficiency (the complemented enzyme is much less active than the native form) and imprecision (resonance energy transfer is a more exacting measure of proximity). Thus, live animal BRET assays would be a useful development and are described in a recent paper from De and Gambhir,. These authors dispense with the traditional method of imaging BRET with a microplate reader and show that a cooled CCD camera can be adapted to pick up BRET signal instead. Like the traditional microplate reader, the CCD camera can pick up BRET signals for multiwell plates of lysed or living cells. However, the same system can also be used for measurement of BRET in nude mice. The authors demonstrate proof-of-principle by confirming that they can detect the rapamycin-dependent interaction of FKBP and FRAP. Although BRET signal could be detected in both superficial and deep tissue, the BRET ratio (an interaction measure) could not be calculated when the signal originated from a tissue depth of more than a few millimeters. Even so, with further optimization, the approach appears applicable for screening drugs that mediate or block clinically relevant protein interactions. -ND

De and Gambhir. 2005. Noninvasive imaging of protein-protein interactions from live cells and living subjects using bioluminescent resonance energy transfer. The FASEB Journal 10.196/fj.05-4628fje.

Both Rhyme and Reason

Attempts to increase the throughput in protein expression experiments using the large number of genes identified by complete genome sequencing have only been moderately successful. Current techniques involve singly cloning and expressing genes of interest, followed by scale-up of cultures and large-scale purification. Solubility problems, particularly in bacterial cultures overexpressing foreign protein, continue to be an issue, while the cost, time, and effort required for this type of work is often prohibitively high. To try to solve some of these issues, a new methodology, called pooled ORF expression technology (POET) has been developed through the collaboration of a number of groups at the NIH, Uniformed Services University, and Dana-Farber Cancer Institute. The protocol attempts to take some of the guess-work out of expression studies by predicting which pools of cloned transcripts will produce practical yields of soluble proteins. A library of ORFs is first subcloned into an expression vector carrying a hexahistidine affinity tag and transformed into a bacterial host. Expressed proteins are purified using the tag, and their abundance is determined by 2-D gel electrophoresis, with those proteins producing the most intense spotting assumed to be both highly expressed and soluble. Individual proteins can then be identified by mass spectrometry, and their individual parental clone expressed at high levels. The procedure was tested using the Caenorhabditis elegans ORFeome and shown to be capable of identifying positive clones that highly expressed soluble proteins, while negative clones (not identified on the 2-D gel) showed little expression. Accepting that some inherent limitations exist and that none of the techniques used are new or necessarily straightforward to perform, in combination they create a useful protocol for large-scale screening of ORFeomes with a significant time and cost saving, predicted by the authors to be between 10-and 100-fold. -SS

Gillette et al. 2005. Pooled ORF expression technology (POET): using proteomics to screen pools of open reading frames for protein expression. Molecular & Cellular Proteomics 4(11):1647-1652.