Application Notes

4 APPLICATION NOTE ©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. In a second set of experiments, we aimed to recapitulate the stabilizing effects of Ca 2+ -Ions on both α-amylase isoforms. Ca 2+ -ions have previously been shown to be required for increased T m s of different amylase isoforms, ranging from virtually no effect for Alteromonas amylase to an increase of T m by 50 °C for Bacillus licheniformis amylase [9]. In order to study the effects of Ca 2+ -ions on PPA and TAKA stability, we incubated both proteins in buffer with 5 mM EDTA to remove bound Ca 2+ for 30 minutes prior to thermal unfolding experiments. As expected, removal of Ca 2+ -ions by EDTA resulted in a marked decrease in T m for both amylase isoforms (Figure 4). Changes to this T m were more pronounced for PPA (16.6 °C) than it was for TAKA (12 °C), which correlates nicely with previously published results (PPA 17 °C, TAKA 14 °C) [10, 11]. Effects of buffer additives on amylase thermal stability A screening for additives and buffer conditions that improve protein stability, also referred to as formulation screening, is of key importance to achieve maximal shelf life of antibodies and other biologicals. Using Prometheus, we tested the effects of different buffer additives that were previously shown to increase protein stability, namely glycerol, sucrose, trehalose and sorbitol, at concentrations ranging from 10 % to 40 % (w/v), on PPA and TAKA. The formulation screen of 16 different buffer conditions for each amylase isoform was performed in a single run, with a temperature range from 20 °C to 90 °C and a shi of the fluorescence emission maximum towards higher wavelengths ("redshi ") or lower wavelengths ("blueshi "). Thermal unfolding of PPA and TAKA was performed at a heating rate of 1 °C/minute, which allows for a precise determination of the onset of protein unfolding as well as for precise fitting of the folded- unfolded transition by mathematical models. Figure 2 shows the changes of tryptophan fluorescence of PPA and TAKA upon thermal unfolding. Notably, for TAKA, the raw fluorescence data from both wavelengths show a clear transition from folded to unfolded (Figure 2A, le ) which could be directly used for T m analysis. In contrast, this transition is not evident from the raw data for PPA (Figure 2B, le ). Moreover, while TAKA displayed a typical unfolding profile with a shi of the tryptophan fluorescence towards higher wavelengths (redshi ), PPA showed a less common shi of the tryptophan fluorescence towards lower wavelengths (blueshi ). The standard deviation of the results shown in the table in Figure 3B from the first derivative analysis is small, demonstrating the maximal reproducibility of the results. Thus, the Prometheus NT.48 can be used to precisely determine T m values with a minimum of sample and time consumption. The results show that both, reproducibility and precision of thermal unfolding experiments with PPA and TAKA were very high (Figure 3A and B). The obtained T m values closely matched reported values from the literature [9].

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