Cell-free protein synthesis of membrane (1,3)-β-d-glucan (curdlan) synthase: co-translational insertion in liposomes and reconstitution in nanodiscs

Biochim Biophys Acta. 2013 Feb;1828(2):743-57. doi: 10.1016/j.bbamem.2012.10.003. Epub 2012 Oct 12.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Agrobacterium / metabolism
  • Catalysis
  • Cell-Free System
  • Escherichia coli / metabolism
  • Glucose / chemistry
  • Glucosyltransferases / chemistry*
  • Liposomes / chemistry*
  • Microscopy, Electron, Transmission / methods
  • Nanoparticles / chemistry*
  • Nanotechnology / methods*
  • Peptides / chemistry
  • Plasmids / metabolism
  • Protein Biosynthesis
  • Proteins / chemistry
  • RNA, Messenger / metabolism
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Surface-Active Agents / chemistry
  • Trypsin / chemistry
  • Uridine Diphosphate / chemistry
  • beta-Glucans / chemistry*


  • Liposomes
  • Peptides
  • Proteins
  • RNA, Messenger
  • Surface-Active Agents
  • beta-Glucans
  • Uridine Diphosphate
  • curdlan
  • Glucosyltransferases
  • 1,3-beta-glucan synthase
  • Trypsin
  • Glucose