A generalizable protocol for expression and purification of membrane-bound bacterial phosphoglycosyl transferases in liponanoparticles.

Abstract

Phosphoglycosyl transferases (PGTs) are among the first membrane-bound enzymes involved in the biosynthesis of bacterial glycoconjugates. Robust expression and purification protocols for an abundant subfamily of PGTs remains lacking. Recent advancements in detergent-free methods for membrane protein solubilization open the door for purification of difficult membrane proteins directly from cell membranes into native-like liponanoparticles. By leveraging autoinduction, in vivo SUMO tag cleavage, styrene maleic acid co-polymer liponanoparticles (SMALPs), and Strep-Tag purification, we have established a robust workflow for expression and purification of previously unobtainable PGTs. The material generated from this workflow is extremely pure and can be directly visualized by Cryogenic Electron Microscopy (CryoEM). The methods presented here promise to be generalizable to additional membrane proteins recombinantly expressed in E. coli and should be of interest to the greater membrane proteomics community. HighlightsO_LIExpression and purification of full-length Lg-PGTs has proven challenging. C_LIO_LIAutoinduction and in vivo Ulp1 cleavage produces active full-length Lg-PGTs. C_LIO_LISMA and DIBMA are vastly superior to DDM for Lg-PGT solubilization. C_LIO_LIStrep-tag purification yields SMALPs suitable for CryoEM characterization. C_LI