Enzyme‐mediated assembly of chimeric membrane proteins at phospholipid bilayers

Abstract

Integral membrane proteins (IMPs) pose a challenge to study in vitro, as it is difficult to reproduce the membrane-imbedded context of their native state. In this study, we developed a generalized strategy for the assembly of chimeric IMPs within a phospholipid bilayer surface based on a two-step process that first imbeds the insoluble domain of the IMP into a phospholipid layer and then ligates the soluble part of the IMP to the imbedded portion under mild biochemical conditions using a mutant sortase A enzyme. The approach is demonstrated using the transmembrane domain of epidermal growth factor receptor (EGFR) and the soluble extracellular loop of the B-lymphocyte antigen CD20 protein in a POPC tethered bilayer membrane (tBLM). The conditions of the enzymatic reaction were optimized for peptide ligation at the tBLM surface and the role of Ca 2+ ions in ligation efficiency examined. Additionally, binding of the CD20/EGFR chimera in the context of a membrane environment by the rituximab antibody was measured to assess functionality. KEYWORDS CD20, EIS, rituximab, sortase A, SPR, TM domain of epidermal growth factorThis is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.