Cell-free synthesis of a transmembrane mechanosensitive channel protein into a hybrid-supported lipid bilayer

Zachary A. Manzer, Surajit Ghosh, Miranda L. Jacobs, Srinivasan Krishnan, Warren R. Zipfel, Miguel Pineros, Neha P. Kamat, Susan Daniel*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


Supported lipid bilayers (SLBs) hold tremendous promise as cellular-mimetic structures that can be readily interfaced with analytical and screening tools. The incorporation of transmembrane proteins, a key component in biological membranes, is a significant challenge that has limited the capacity of SLBs to be used for a variety of biotechnological applications. Here, we report an approach using a cell-free expression system for the cotranslational insertion of membrane proteins into hybrid-supported lipid bilayers (HSLBs) containing phospholipids and diblock copolymers. We use cell-free expression techniques and a model transmembrane protein, the large conductance mechanosensitive channel (MscL), to demonstrate two routes to integrate a channel protein into a HSLB. We show that HSLBs can be assembled with integrated membrane proteins by either cotranslational integration of protein into hybrid vesicles, followed by fusion of these proteoliposomes to form a HSLB, or preformation of a HSLB followed by the cell-free synthesis of the protein directly into the HSLB. Both approaches lead to the assembly of HSLBs with oriented proteins. Notably, using single-particle tracking, we find that the presence of diblock copolymers facilitates membrane protein mobility in the HSLBs, a critical feature that has been difficult to achieve in pure lipid SLBs. The approach presented here to integrate membrane proteins directly into preformed HSLBs using cell-free cotranslational insertion is an important step toward enabling many biotechnology applications, including biosensing, drug screening, and material platforms requiring cell membrane-like interfaces that bring together the abiotic and biotic worlds and rely on transmembrane proteins as transduction elements.

Original languageEnglish (US)
JournalACS Applied Bio Materials
StateAccepted/In press - 2021


  • Cell-free protein synthesis
  • Diblock copolymers
  • Hybrid vesicle
  • Lipids
  • Supported lipid bilayer
  • Transmembrane proteins

ASJC Scopus subject areas

  • Biomaterials
  • Chemistry(all)
  • Biomedical Engineering
  • Biochemistry, medical

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