Application Notes

Microscale Thermophoresis measurements on in vitro synthesized proteins

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2 NMR they all have their pitfalls. SPR requires a highly pure sample and a successful and time consuming immobilization strategy. ITC requires vast amounts of material that can be a challenge to produce at sufficient high quality. The present study was designed to show that the novel and powerful technology MST used in combination with cell-free protein biosynthesis is a very efficient and smart shortcut to this classical approach to obtain quantitative protein interaction data. We have selected two interactions to demonstrate this possibility. Calmodulin and its interaction with an M13 peptide fused to citrine was analyzed in a first set of experiments, and as a second model system we used Cyclophilin A and a single chain antibody fragment directed against it. Results Calmodulin was synthesized with a short leader sequence containing an amber codon, which allowed the site-directed introduction of the chemoselective reactive unnatural amino acid p- azido-Phenylalanine by expression in RiNAs RF1- depleted cell-free orthogonal protein translation system. Fig. 2 SDS-PAGE analysis of in vitro synthesized and dye- labelled Calmodulin and its interaction partner M13-Citrine. Left panel: Coomassie stained gel; right panel: Fluorescence Image (excitation 633 nm). Lane 1: M13-Citrine, lane 2: CaM, lane 3: CaM of lane 2 modified with DyLight650, lane 4: 0.75 µg BSA as a standard. 1.5 µl reaction each were loaded in lanes 1, 2 and 3. Positions of synthesized proteins are marked with asterisks (*). Full conversion of synthesized CaM with dye is indicated by shifting to higher molecular weight (compare lane 2 and lane 3). This amino acid was then selectively reacted with a fluorescent dye by means of Staudinger ligation, providing the labeled molecule for the MST analysis. The unlabeled interaction partner M13- Citrine (M13 peptide fused to Citrine) was expressed similarly. In order to first assess the quality of the in vitro synthezied Calmodulin we tested its binding to Ca 2+ ions. Previously in a purified system we had shown an affinity of 2.8 µM, and in this set of experiment we determined an affinity of 4.14 µM. Fig. 3 Binding of Ca 2+ to Calmodulin. The K d was determined to be 4.14 ± 2.26 µM. Inset displays the original normalized MST traces. In a second step we titrated the synthesis reaction of cell-free synthesized M13 peptide as a fusion protein with citrine, named M13-Citrine, without any purification step to Calmodulin, and performed a further MST measurement. The affinity was determined to be 5 nM. This corresponds nicely with the literature value of 1 nM for the free M13 peptide (Blumenthal et al. 1985). Fig. 4 Binding of M13-Citrine to Calmodulin (black dots). The affinity was determined to a K d = 4.48 ± 1.77 nM. As a control a non-binding mutant of M13-Citrine was used (red dots). No binding was observed. Inset displays the original normalized MST traces of the experiment using Calmodulin and M13- Citrine. In a control experiment the synthesis reaction of a non-binding M13-Citrin mutant was used. An identical titration series has been prepared and analyzed in the same way as the M13 (wt)-Citrin interaction by MST. The corresponding analysis of the experiment showed no binding at all, as it was expected. As a second interaction we investigated the one between Cyclophilin A and a single chain antibody fragment named AntiEC5218 directed against it. 72- 55- 36- 28- 17- 10- * * * 1 2 3 4 1 2 3 4

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