Issue link: https://resources.nanotempertech.com/i/1384283
TECHNICAL NOTE 6 ©2021 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. Conclusion The goal of researchers working in protein production is not just to produce enough protein, but to produce a protein that is also properly folded and functional. SDS-PAGE has been used as an inexpensive method for relative comparison of total protein levels for decades, but it doesn't offer information on the folding state of the target protein. A comprehensive expression screening approach must consider protein conformational stability besides high expression levels — particularly for target proteins destined for affinity screening or structural studies. In this case study, SDS-PAGE and Andromeda are used to monitor the effects of growth temperature, growth medium, and expression host strain on the expression levels of His- tagged HRV3C protease, and the information provided from each method is compared. With Andromeda, it was possible to quickly identify conditions that resulted in high expression levels of properly folded recombinant His-tagged HRV3C. Where SDS-PAGE showed higher expression levels, Andromeda revealed that the additional amount was potentially not properly folded (growth temperature screen). And where SDS-PAGE showed no difference in expression level, Andromeda clearly indicated a condition that resulted in a higher amount of stable protein (host strain screen). From these experiments, it is clear that Andromeda provides information about the recombinant proteins that SDS-PAGE doesn't, and therefore complements other methods used in expression screening. With its fluorescence-based approach for thermal stability analysis of His-tagged or GFP- fused proteins even in crude mixtures like lysate, Andromeda allows for rapid expression level screening while at the same time providing valuable additional information on protein stability and folding state. If small molecule ligands are available, Andromeda performs quick thermal shi assays confirming functionality directly in cell lysate — making it an ideal tool for finding the optimum expression conditions for recombinant protein targets destined to downstream applications such as drug discovery or structural biology. Andromeda delivers additional stability information in a rapid and straightforward approach with minimal assay setup directly from cell lysate, so researchers can assess