Technical Notes

TECHNICAL NOTE 3 ©2021 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. Results Monitoring growth temperature Choosing the correct growth temperature is crucial for optimum protein expression. The right trade-off between biomass growth and protein thermal stability must be determined empirically for each protein target individually, as conditions that may favor more biomass and therefore more total protein may also lead to accumulation of misfolded protein in inclusion bodies. To determine optimum growth temperatures for His- tagged HRV3C, identical E. coli cultures expressing the protein were grown at 16 °C, 20 °C, and 25 °C. When clarified lysates are analyzed by SDS-PAGE, the results indicate that the higher the temperature, the higher the total yield of His-tagged HRV3C, as suggested by the increased intensity of the Coomassie blue stain in the bands corresponding to His-tagged HRV3C (Figure 2A). Analysis of the clarified lysates with Andromeda shows that all three growth temperatures produce properly folded recombinant protein, indicated by the presence of peaks in the first derivative plot (Figure 2B). However, the largest peak is seen for 16 °C, an indication that this temperature may yield the highest amount of properly folded protein amongst all three temperatures tested. Even though the overall yield is highest at 25 °C, the data from Andromeda suggests that optimum growth temperature that results in the expression of more stable protein for this system is 16 °C L 10 15 20 25 37 50 75 C 16 °C 20 °C 25 °C P L C P L C P First Derivative Temperature [°C] 30 35 40 45 50 55 60 16°C 20°C 25°C Figure 2: Samples collected from cultures grown at three different temperatures were analyzed via SDS-PAGE and Andromeda. A) SDS-PAGE of the lysate (L), clarified lysate (C), and purified protein (P). For easier comparison with subsequent Andromeda analysis, the lanes showing clarified lysate samples are highlighted in boxes. The expected molecular weight of the protein of interest is indicated by an arrow next to the standard. B) Andromeda unfolding profiles of clarified lysate samples collected from cultures grown at 16 °C (yellow), 20 °C (purple), and 25 °C (blue). Unfolding profiles represent the average of at least three technical replicates. The shaded area indicates the standard deviation amongst all technical replicates. A B

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