Profiling Protein Interactions
Researchers at Merrimack Pharmaceuticals are exploring the signaling pathways activated by the fusion protein Etv6-NTRK3, formed from a combination of the Etv6 transcription factor and the tyrosine kinase domain of NTRK3. The fusion gene is present in a variety of tumor types. Tyrosine phosphorylation can lead to protein activation and subsequent downstream-signaling activity. Merrimack scientists designed a system to determine which tyrosine moities in Etv6-NTRK3 are phosphorylated and which are required for oncogenic transformation.
Jack Allen, a scientist at Merrimack, described the group’s use of tandem mass spectrometry to identify 17 sites of tyrosine phosphorylation on the fusion protein. They then screened each site using protein domain microarrays printed onto glass slides that represent nearly all of the human Src Homology 2 and phospho-tyrosine binding domains to pinpoint protein-interaction events that could trigger signaling. Allen reported on the use of site-directed mutagenesis in combination with phenotypic assays to identify a site on the Etv6 component of the protein that is required for cellular transformation in vitro and in vivo.
Andrea Weston, senior research investigator at BMS, spoke about high-throughput screening with real-time PCR. In high-throughput screening, quantitative RT-PCR can be used to profile the effects of small molecules on gene expression in a real-life context. Although not a new technology, RT-PCR has not been readily amenable to high-throughput processes, but strategies to enable this are now available.
Challenges have included the laborious task of isolating RNA while preventing its degradation (cell lysis buffers now allow for qRT-PCR to be done directly in cell lysates); the two-step process and cDNA intermediate step required with traditional PCR methods (replaced by one-step PCR protocols that are readily automatable and require no mixing); the relatively high cost of PCR reagents and the time factor for thermal cycling (being overcome by miniaturization and the evolution to 1,536-well formats); and the fact that any error in liquid transfer will be amplified during the PCR reaction (a problem minimized by state-of-the-art low-volume, precise acoustic dispensing technologies).
Using high density plate formats and acoustic dispensing, reaction volumes have decreased to as low as 0.5 µL and 1,536 different experiments can be done in a single run. Weston presented data comparing RT-PCR performed in cell lysates with amplification of isolated RNA and described the results as “encouraging”—though not as clean as with isolated RNA—adding, “you do lose dynamic range.”
She described acoustic dispensing technology from Labcyte (Echo® liquid handlers and software) and EDC Biosystems (ATS Acoustic Transfer System), as well as Beckman Coulter’s microfluidic system (BioRAPTR® FRD®), each with advantages and disadvantages such as fixed versus variable droplet sizes, greater or lesser dead volume, and software-defined or user-calibrated adjustments to compensate for variability in the meniscus.
Russell presented preliminary work using acoustic transfer of cell lysates from 1,536-well plates, with early feasibility testing yielding “promising” results with both the Labcyte and EDC instruments. As acoustic transfer drives down volumes, the cost of doing RT-PCR decreases. Weston hopes that competition will push the cost down to no more than $0.10/well. BMS is working with Roche to develop a fully automated PCR process based on the LightCycler® 1536 instrument.