Sortase A-Mediated Farnesylation of Cdc42 In Vitro

Cdc42, a Rho-family GTPase, plays a pivotal role in establishing polarity in Saccharomyces cerevisiae by accumulating on the membrane at the site of bud emergence. Cdc42's ability to bind to membranes, mediated by prenylation, is essential for its function. Prenylation involves either the post-translational addition of a 15-carbon farnesyl group or a 20-carbon geranylgeranyl group to Cdc42's C-terminus. One of the major challenges in studying the physical and chemical interactions of Cdc42 at the polarity spot in vitro is obtaining prenylated Cdc42. Here, we present a streamlined, sortase A-based approach to farnesylate Cdc42 in vitro. This method uses Escherichia coli-expressed Cdc42 with a sortase A recognition motif, facilitating efficient farnesylation and purification using a size exclusion-based strategy. The farnesylated Cdc42 retains functionality, as evidenced by membrane binding and by its GEF-regulatory GTPase activity, making it suitable for further biophysical and biochemical investigations.