Eukaryotic cell-free synthesis was used to incorporate the large and complex multispan plant membrane transporter Botl in a functional, form into a tethered bilayer lipid membrane. The electrical properties of the protein-functionalized tethered bilayer were measured using electrochemical impedance spectroscopy and revealed a pH-dependent transport of borate ions through the protein. The efficacy of the protein synthesis has been evaluated using iminunoblot analysis.