Sortase A-mediated farnesylation of a protein in vitro v1

This protocol describes a Sortase A-based approach to farnesylate a protein of interest (POI) in vitro. This method leverages E. coli-expressed protein with a Sortase A recognition motif, facilitating efficient and accessible farnesylation and purification using a purification tag-based strategy. In brief: E. coli expression system are used to produce a double-tagged protein construct with the Sortase recognition motif flanked with two purification tags (C-terminal His-tag and another other N-terminal tag) [Tschirpke et al., 2023]. In the labeling reaction, commercially available Sortase A enzyme is used to cleave the C-terminal His-tag off the POI and in one step ligate triclycine cysteamine farnsyl [Tschirpke et al., 2026] to the C-terminus of the protein. The reaction mixture is cleaned by size exclusion chromatography (SEC), optionally followed by His-affinity chromatography (His-AC), to isolate the farnesylated POI. Background: Transpeptidase Sortase A, derived from S. aureus, can label proteins in a precise site-specific manner due to its ability to perform both cleavage and ligation in one step. Sortase A recognizes a LPXTG motif (where L is leucine, P is proline, X is a variable amino acid, T is threonine, and G is glycine) in a substrate, cleaves the bond between threonine and glycine in the motif, and subsequently catalyzes the formation of an amide bond between the threonine of the substrate and the N-terminal glycine of a compatible probe [Antos et al., 2017; Guimaraes et al., 2013]. Sortase A has almost no requirements for the structure or sequence of the probe as long as it contains oligoglycine at the N-terminus. Sortase A has been used for many different labeling purposes ranging from labeling with fluorescent dyes, nucleic acids, or lipid chains as well as protein-protein and protein-membrane conjugations both in vivo and in vitro [Pritz et al., 2007; Antos et al., 2008; Sakamoto et al., 2010; Chen et al., 2016; Vilen et al., 2023]. In this protocol we utilize a Sortase A-mediated reaction for the farnesylation of a POI in vitro.