Technical Notes

TECHNICAL NOTE 2 ©2021 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. His-tagged HRV3C expression levels were monitored while systematically screening for optimum growth and expression parameters such as growth temperature, growth medium, and expression host strain. The results show that while certain expression conditions yield high expression levels as determined by SDS-PAGE, they may result in proteins with less-than-optimal stability and conformational integrity. Experiments to confirm that the optimal conditions identified with Andromeda result in higher amount of stable purified protein are not shown here since they were outside of the scope of this study. Technology and approach With Andromeda, protein expression and production teams can use the thermal stability characterization of proteins in crude cell lysate to determine their relative abundance and thermal stability. The recombinant protein is detected either via GFP- or His-tag, which can be fluorescently labeled directly in lysate in a one-step process. Changes in fluorescence intensity of the labeled protein are monitored during a thermal ramp to derive unfolding profiles — a fast process that takes only 11 minutes for up to 48 samples. A er the automated derivative analysis, the presence of a peak indicates folded target protein in the sample, while the height of the peak correlates with the amount of folded target protein and serves as an estimate for the yield of folded protein that can be expected a er purification (Figure 1). Andromeda is especially useful for challenging situations such as membrane protein expression, where expression yields are low, and proteins are prone to misfolding. For this study, His-tagged HRV3C expressed in E.coli was labeled directly in clarified lysates with a fluorophore coupled to a tris-NTA moiety that binds with high specificity to the His-tag. First derivative Temperature High expression of folded protein Lower expression of folded protein No expression or completely unfolded protein Figure 1: Schematic of Andromeda protein unfolding profiles. Changes in fluorescence intensity are monitored during a thermal ramp, and the first derivative analysis of the data results in peaks. High peaks (light blue sample) indicate high amounts of folded protein compared to smaller peaks (purple sample). An absence of peaks (dark blue sample) indicates that no protein was detected, or that all target protein is unfolded and therefore likely nonfunctional.

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