Open AccessEnzyme‐mediated assembly of chimeric membrane proteins at phospholipid bilayers
Author(s) -
Scott D.,
Tullman J.,
Kelman Z.,
Marino J.,
Silin V.
Publication year - 2019
Publication title -
engineering reports
Language(s) - English
Resource type - Journals
ISSN - 2577-8196
DOI - 10.1002/eng2.12040
Subject(s) - chemistry , phospholipid , biophysics , lipid bilayer , bilayer , membrane , membrane protein , integral membrane protein , nanodisc , transmembrane protein , biochemistry , popc , context (archaeology) , biology , receptor , paleontology
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.