Cdc42 construct design for in vitro studies

Biological systems are complex by nature. To shine light on their inner workings, the multi-faceted lens of interdisciplinary research is required. One lens is the in vitro approach, in which the physical and biochemical properties and interactions of isolated components are investigated in detail. If the components are proteins, they need to be purified. This is generally done by attaching an N- or C-terminal purification tag to the protein of interest. The rational behind the placement of purification tags and their effect on the protein’s properties is rarely discussed, making in vitro studies less accessible to non-biochemists. Here, we explore the effect of protein construct design and purification tags on the S. cerevisiae protein Cdc42. Cdc42 is an essential small GTPase and the main regulator of polarity establishment and cell division in budding yeast. It’s part of a complex polarity protein network and highly regulated, making it an attractive target for in vitro studies. We show that the T7 lead is a requirement for the Cdc42 expression in the E. coli expression system and that purification tags can influence the expression and degradation levels of Cdc42-sfGFP and Cdc42-mNeonGreen sandwich fusions. Cdc42's GTPase activity, interaction with the GEF Cdc24 and scaffold Bem1 are largely unaffected by Cdc42's N- and C-terminal purification tags. The exception is Cdc42 tagged with an N-terminal Twin-Strep-tag, which shows precipitation issues and a decreased GTPase activity and Cdc24 interaction. We close with using the case of Cdc42 as an example for discussing criteria relevant for protein construct design in general.