Expression and Purification of Integral Membrane Enzymes for Functional and Structural Studies.

Enzymes that reside in the membrane are responsible for a myriad of vital tasks such as assembly of and changes to the membrane itself, and modification of proteins that reside therein or are secreted through the various cellular compartments. Understanding how these enzymes work at a molecular level is most often dependent on high resolution structural information. While the rates of success for the determination of structures of integral membrane proteins in general, enzymes included, have improved considerably-most notably because of recent advances in single particle cryo-electron microscopy (cryo-EM) technology-considerable challenges to obtaining high resolution structures remain. Without a doubt, the quality of the protein sample is the most critical parameter, where a suitable target must first be identified, then expression and purification conditions optimized. Here, we describe in detail a standard approach and workflow, using the prokaryotic glycosyltransferase GtrB as an example. Together, the methods described here demonstrate that careful selection via thorough screening, and optimization of expression and purification conditions, are an invaluable investment of time, ultimately yielding the best target not only for successful high-resolution structural analyses-X-ray crystallography and cryogenic electron microscopy in this case-but also for biochemical and biophysical studies.