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 Ca2+ 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.
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