Review of Technology

Receptor Tyrosine Kinases (RTKs) comprise a large family of single-pass transmembrane proteins. Test-tube phosphorylation assays routinely employ cloned RTK catalytic domain fragments. These catalytic fragments, however, are removed from their membranous environment and thus cannot exhibit full function. The Biological Relevance of results obtained from assays of this type will always be questionable. Consequently, we developed a protocol (patent pending) for template-directed self-assembly of RTK catalytic domains; recreating the membrane environment with the objective of restoring native activity and function. See the flash animation.

What we have here goes well beyond kinase activity - we believe our technology can and will alter the way signaling assays are performed.  Most importantly, we think that results attained using templated protein reagents will be more biologically relevant than those from standard solution-based assays. This will be very useful in screening assays where we expect similar results will be observed in subsequent cell-based assays. Ultimately, we foresee this process becoming a novel platform for drug discovery; properly assembled key components of disease related pathways can be screened for new types of drugs aimed at specific pathway branches.

We also expect that our technology will be useful beyond receptor tyrosine kinases. Any transmembrane protein, including multi-pass transmembrane proteins, as well as any membrane-associated protein that requires oligomerization or complex assembly, is a potential candidate for our platform.

Specifically, we believe we can significantly improve the performance of a number of currently marketed products as well as development of fragments purposefully engineered for use with this templating technology. We have observed significant enhancement of signal for several commercially available products thus far. A brief summary of uses below should make it apparent that our technology will significantly improve the way in vitro signaling assays are performed.

PA Tech’s Technology

Enables assembly of biologically relevant protein units

• With our templating technology, we generate a membrane-associated oligomerized unit that faithfully reproduces the in vivo assembled unit.

• This is not simply induced homo-dimerization of proteins that is achieved by introduction of metals, nor is it induced aggregation of proteins (i.e. using ammonium sulfate or polyethylene glycol to aggregate proteins), our template provides a natural scaffold for assembly of a biologically relevant, catalytic, unit that is capable of recruiting downstream proteins.

Enables “Team Assembly”

• Using our membrane-associated unit of proteins, you can assemble entire teams of proteins (i.e. accessory factors, adaptor proteins, phosphatases, cytoplasmic kinases…) for research purposes or for sale in pathway specific screening kits. See Flash Animation

From template directed assembly to cell-based assays

• Based on our early research and from results with commercially available reagents, we believe that results obtained from inhibitor screens using our template-assembled proteins will more reliably predict the outcome of subsequent cell-based assays.

Save money by using less protein in each assay

• Activity from templated “protein teams” can be significantly higher than with untemplated proteins, enabling us to use less protein while obtaining similar signal to noise in standard phosphorylation assays.

Our technology has potential application in reagent production

• Our templated “protein teams” should be useful in the generation of site-specific phosphorylated enzymes that can be sold as-is or in kits containing pathway components.

A novel drug discovery tool

• Our template-assembled “protein teams” can be used to screen for pathway inhibitors.

Simple, one-reagent addition to mix-n-read assays

• To date, there are no indications that our template will interfere with antibody based detection or fluorescence based assays commonly used in high-throughput assays.

Not limited to RTK’s

• We expect this technology to be applicable to other signaling elements, such as a specific loop from a multi-pass protein that is known to recruit or activate another protein with recruited factors, so that these elements can now be assembled and presented as if on a cell surface.