Application Notes

Microscale Thermophoresis measurements on in vitro synthesized proteins

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1 Protein-Protein Interaction Analysis Application Note NT-MO-013 MicroScale Thermophoresis Measurements on in vitro Synthesized Proteins Michael Gerrits 1 and Jan Griesbach 2 1 RiNA GmbH, Berlin, Germany ( 2 NanoTemper Technologies GmbH, Munich, Germany ( Abstract Nowadays, especially through the advent of systems biology, and the ability to generate predictive models, it is even more important to transform biology from a qualitative to a quantitative science. Interactions between proteins not only need to be identified, but their equilibrium rate constants need to be determined as well. Here we describe a very elegant and simple system to obtain quantitative data for protein-protein interactions using cell-free protein biosynthesis in combination with MicroScale Thermophoresis. We have been able to characterize the interaction of Calmodulin with Ca 2+ as well as its ligand M13 straight in the in vitro synthesis reaction. Furthermore, we are demonstrating the generic applicability of this approach with another set of proteins, an Antibody fragment and Cyclophilin A. All this work has been done without tedious expression and prior purification of the interacting proteins. Introduction It is crucial in Biology these days to provide not only qualitative data, but also quantitative data. Now, demonstrating an interaction is fairly simple and routinely done by high throughput methods such as yeast-two-hybrid or pull down assays, and MS. However, transforming this knowledge into quantitative data to feed into predictive models of regulatory networks of the cell is far from easy. Usually the process starts with the careful selection of the two interacting proteins, the right iso- or spliceforms and the selection of the appropriate domains. These have to be passed through construct design, as a lot of mammalian full length proteins are not readily expressed in a heterologous system. Expressing these proteins is as well not easy and requires at least a laboratory with S1 biohazard safety standards. After being able to express the protein it still needs to be purified. Fig. 1 In vitro expression and labeling scheme! Protein samples can be used immediately after without purification for the synthesis mix. Purification of biomolecules for biophysical analysis requires high quality samples and a high degree of purification which is rarely achieved by a single affinity purification step. If one looks now at the long time standard methods like SPR, ITC or

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