Sortase A-mediated farnesylation of Cdc42in vitro

Abstract 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 mayor challenges in studying the biophysical and biochemical interactions of Cdc42 at the polarity spot in vitro is obtaining prenylated Cdc42, due to labor-intensive and not easily reproducible traditional methods. Here, we present a streamlined, Sortase A-based approach to farnesylate Cdc42 in vitro. This method leverages E. coli-expressed Cdc42 with a Sortase A recognition motif, facilitating efficient and accessible farnesylation and purification using a purification tag-based strategy. The farnesylated Cdc42 retains functionality, as evidenced by GTP-dependent membrane binding, making it suitable for further biophysical and biochemical investigations. Additionally, our method can be easily adapted to yield geranyl-geranylated Cdc42. Competing Interest Statement The authors have declared no competing interest. Abbreviations - Boc - tert-butoxycarbonyl protecting group - Gly - glycine - DCM - dichloromethane - DMF - N,N-dimethylformamide - PyBOP - benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate - DiPEA - N,N-diisopropylethylamine - TFA - trifluoroacetic acid - MeOH - methanol - NMR - nuclear magnetic resonance - DMSO - dimethylsulfoxide