A membrane-embedded curdlan synthase (CrdS) from Agrobacterium is believed to catalyse a repetitive addition of glucosyl residues from UDP-glucose to produce the (1,3)-β-d-glucan (curdlan) polymer. We report wheat germ cell-free protein synthesis (WG-CFPS) of full-length CrdS containing a 6xHis affinity tag and either Factor Xa or Tobacco Etch Virus proteolytic sites, using a variety of hydrophobic membrane-mimicking environments. Full-length CrdS was synthesised with no variations in primary structure, following analysis of tryptic fragments by MALDI-TOF/TOF Mass Spectrometry. Preparative scale WG-CFPS in dialysis mode with Brij-58 yielded CrdS in mg/ml quantities. Analysis of structural and functional properties of CrdS during protein synthesis showed that CrdS was co-translationally inserted in DMPC liposomes during WG-CFPS, and these liposomes could be purified in a single step by density gradient floatation. Incorporated CrdS exhibited a random orientation topology. Following affinity purification of CrdS, the protein was reconstituted in nanodiscs with Escherichia coli lipids or POPC and a membrane scaffold protein MSP1E3D1. CrdS nanodiscs were characterised by small-angle X-ray scattering using synchrotron radiation and the data obtained were consistent with insertion of CrdS into bilayers. We found CrdS synthesised in the presence of the Ac-AAAAAAD surfactant peptide or co-translationally inserted in liposomes made from E. coli lipids to be catalytically competent. Conversely, CrdS synthesised with only Brij-58 was inactive. Our findings pave the way for future structural studies of this industrially important catalytic membrane protein.

Eukaryotic cell-free synthesis was used to incorporate the large and complex multispan plant membrane transporter Bot1 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 immunoblot analysis.

Membrane proteins control fundamental processes that are inherent to nearly all forms of life such as transport of molecules, catalysis, signaling, vesicle fusion, sensing of chemical and physical stimuli from the environment, and cell-cell interactions. Membrane proteins are harbored within a non-equilibrium fluid-like environment of biological membranes that separate cellular and non-cellular environments, as well as in compartmentalized cellular organelles. One of the classes of membrane proteins that will be specifically treated in this article are transport proteins of plant origin, that facilitate material and energy transfer at the membrane boundaries. These proteins import essential nutrients, export cellular metabolites, maintain ionic and osmotic equilibriums and mediate signal transduction. The aim of this article is to report on the progress of membrane protein functional and structural relationships, with a focus on producing stable and functional proteins suitable for structural and biophysical studies. We interlink membrane protein production primarily through wheat-germ cell-free protein synthesis (WG-CFPS) with the growing repertoire of membrane mimicking environments in the form of lipids, surfactants, amphipathic surfactant polymers, liposomes and nanodiscs that keep membrane proteins soluble. It is hoped that the advancements in these fields could increase the number of elucidated structures, in particular those of plant membrane proteins, and contribute to bridging of the gap between structures of soluble and membrane proteins, the latter being comparatively low.