Expression, Purification, and Structural Characterization of Membrane Proteins: A Case Study of the ABC Transporter MsbA.

Integral membrane proteins play essential roles in cellular physiology but present formidable challenges in their overexpression, purification, and structural characterization. This chapter outlines a detailed workflow for the expression, purification, and structural analysis of the ABC transporter MsbA from Pseudomonas aeruginosa (PaMsbA). This system exemplifies how biochemical optimization, native mass spectrometry (MS), and cryo-electron microscopy (cryo-EM) can be integrated to investigate membrane protein function and structure. The workflow includes expression and purification of PaMsbA from Escherichia coli for structural and biophysical studies. A unique feature of PaMsbA is its activation by divalent metals (Zn²⁺, Ni²⁺, and Mn²⁺), which is necessary for vanadate trapping and high-resolution cryo-EM structure determination. Together, these approaches establish a framework for preparing high-quality membrane protein samples for mechanistic and structural investigations.