In Vitro Production of Full-Length Myosin 2 and Visualization of Actin Filament Motility by Total Internal Reflection Microscopy.

In vitro protein production constitutes a valid resource to generate data regarding the structure and function of myosin 2. This technique can be combined with proteins, molecular complexes and small molecules to study myosin II conformation, binding to actin and the effect of various myosin and actin modulators. Here, we describe a straightforward protocol to produce and purify NM2 (non-muscle myosin 2) in Spodoptera frugiperda Sf9 cells. We also provide an example of produced protein use in an in vitro actin motility assay using TIRF microscopy to visualize myosin 2-driven F-actin motion and quantify it using ImageJ. This type of analysis can be implemented in various ways and become useful for drug screenings and characterization of novel, disease-causing mutations.