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Life Tech Scientists Develop Protein Thermal Shift Assay for Real-Time PCR Systems

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By Ben Butkus

MILLBRAE, Calif. – Life Technologies has developed and validated a protein thermal shift assay that allows users to conduct various proteomics studies on any Life Tech real-time PCR system, scientists from the company said this week.

The assay has the potential to facilitate drug discovery by measuring protein thermal stability, identifying suitable buffer conditions, and measuring protein-ligand interactions; and could open up a new market for Life Tech's real-time PCR systems.

And even though the assay is in beta-test phase and not yet widely available, early users have already expressed interest in acquiring a PCR system from Life Tech specifically for the protein analysis capabilities, company representative said.

Life Tech scientists Carole Bornarth and Mousumi Rath discussed the new assay in a pair of scientific poster presentations at Intelligent Enterprise Solutions and TATAA Biocenter's US qPCR Symposium, held here this week.

In their presentations, Bornarth and Rath noted that the difficulty and expense involved with isolating or obtaining proteins of interest "demands that care be taken with subsequent handling or storage in order to maximize the utility and longevity of the protein and to ensure data quality is not affected by degradation or aggregation events."

As such, researchers currently use a variety of techniques to interrogate conditions such as salt concentration, pH, specific ligand binding, and temperature, all of which may affect protein stability.

However, many of these techniques "can take days or require a large amount of valuable sample," Rath told PCR Insider. As such, the Life Tech researchers strove to develop an assay for protein stability that would require a "minimum amount of sample and time," Rath added.

Enter the protein thermal shift assay, which was originally called ThermoFluor and was developed at 3-Dimensional Pharmaceuticals, which Johnson & Johnson acquired in 2003. Life Tech now owns exclusive rights to the technology, the company said.

In order to adapt the protein thermal shift assay to real-time PCR systems offered by Life Tech's Applied Biosystems business, the researchers developed a software package called TmTool, written in Matlab, which utilizes the Boltzmann equation to calculate the Tm of the protein from its fluorescence melt plot. Bornarth said that some customers interested in the assay have thought of it as a high-resolution melt analysis application for proteins, although that is a fairly crude comparison.

In addition, the Life Tech scientists use a non-specific dye, such as Sypro Orange, which alleviates any need for prior knowledge of protein function or ligand activity. When the protein of interest starts to denature in response to an increase in temperature, the hydrophobic core of the protein is exposed, and the dye reacts to this change in environment and begins fluorescing, according to the researchers.

In their scientific posters, Bornarth, Rath, and colleagues demonstrated how the PTS assay can identify ligands to co-crystallize a protein of interest, using the example of the YraM lipoprotein encoded by the HI1655 gene, which is crucial to the growth and viability of Haemophilus influenzae.

Not only was the technique "fast and efficient for identifying the optimal conditions" that would lead to successful crystallization; but it also can help provide a "better understanding of the … pathogen and better design of drug therapies to combat this pathogen," the researchers wrote.

The Life Tech scientists also demonstrated how the assay could be used in buffer and ligand screening for T4 DNA ligase; as well as how it can discriminate between point mutation variants of M-MLV, SuperScript II, and SuperScript III reverse transcriptases.

Lastly, the researchers demonstrated how the assay can be used to detect the binding of antibodies to target proteins, which opens up the possibility of its use in drug discovery.

"There is absolutely potential for this in drug screening at pharma companies," Bornarth said. "People are starting to explore using this specifically for this purpose."

The scientists have obtained data from the assay on the entire range of Applied Biosystems real-time PCR platforms, including the 7900 HT, 7500 Fast and StepOnePlus, and recently launched ViiA 7.

In general, the assay requires only a few micrograms of protein to generate accurate results; and can take anywhere from a few hours to most of a day, depending on which real-time PCR platform is used, Bornarth and Rath said.

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