Application Notes

3 Again the fluorescent dye was introduced within a leader sequence at the N-terminus of the antibody fragment, through a similar process as used for Calmodulin. A dilution series of an unlabeled Cyclophilin An in vitro synthesis reaction was prepared and mixed with the labeled AntiEC5218. These samples were loaded in MST standard treated capillaries and mounted in the NanoTemper Monolith. Analysis of the experiment yielded an affinity of 46 nM. Fig. 6 Binding of Cyclophilin A to AntiEC5218. The K d = 46.0 ± 17 nM was derived through curve fitting. Inset displays the original normalized MST traces. Conclusion In this study it was shown that quantitative binding data can be generated in a matter of a few hours using RiNAs in vitro translation systems for the synthesis of fluorescently labeled proteins as well of their unlabelled interaction partners in combination with NanoTempers MicroScale Thermophoresis technology for interaction analysis. Non-purified proteins in whole synthesis reactions can be used after a short desalting step, at which volumes and protein concentrations of the cell-free protein biosynthesis reactions fit the needs of MST very well. Neither elaborate protein purification nor cell culture facilities or expensive HPLC and FPLC equipment are required to prepare interaction partners for the measurement of affinities. Furthermore, the time to obtain these data is minimal. In vitro synthesis reactions take on average a few hours, at most overnight, while the MST experiment itself can be performed in a matter of minutes. The combination of both, allows the synthesis of the desired target proteins and the characterization of their interaction in less than a day. Material and Methods Protein Synthesis and Labeling Protein synthesis was performed using an E. coli derived cell-free translation system depleted from termination factor RF1 (Gerrits et al. 2007, Serwa et al. 2009) and supplemented with enriched fractions of orthogonal amber suppressor tRNA and p-azido phenylalanyl-tRNA synthetase (Chin et al. 2002) specific for p-azido phenylalanine. The system contained p-azido phenylalanine (Bachem) in addition to all 20 natural amino acids. A mixture of reduced and oxidized glutathione was added supplementary for the synthesis of the single chain antibody fragment AntiEC5218 to maintain an oxidizing environment for the formation of disulfide bonds. Following protein synthesis the reactions were desalted two times against PBS using gel filtration spin columns to remove low molecular weight components as non-incorporated amino acids. Aliquots of desalted reactions with the p-azido phenylalanine containing proteins were incubated with fluorescent dye (DyLight650, Pierce, Thermo Scientific) accomplishing Staudinger ligation (Prescher et al. 2005) and desalted using NAP5 columns against PBS. A detailed description of this orthogonal cell-free system and the whole procedure will be published shortly (manuscript in preparation). Further informations about the used systems are available at RiNA GmbH and NanoTemper GmbH. Protein Quantification Protein concentrations were calculated based on a reaction containing radioactively labeled leucine ( 14 C-leucine) that was performed in parallel and subjected to hot TCA precipitation. A BSA standard was used in addition to control the calculated amount of protein via gel electrophoresis and coomassie staining. Assay conditions All synthesis reactions had been gel filtrated against PBS. Calmodulin was kept constant at a concentration of 5 nM in both experiments, while Anti EC5218 was kept at a concentration of 25nM. The ligands, Ca 2+ , M13-Citrine, M13 (mutant)- Citrine and Cyclophilin A were diluted in PBS buffer containing 0.025 % Tween-20 for the dose response experiments. The measurement of the interaction between Calmodulin and M13-Citrine was carried out in the presence of 10 µM CaCl 2 . Instrumentation The measurements were done on a NanoTemper Monolith NT.115 instrument. For the interaction of Calmodulin with Ca 2+ , the measurement was performed at 80 % LED and

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