Cell-free protein synthesis of membrane (1,3)-β-d-glucan (curdlan) synthase: co-translational insertion in liposomes and reconstitution in nanodiscs.
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.
Australian Centre for Plant Functional Genomics, School of Agriculture, Food and Wine, University of Adelaide, Waite Campus, Australia.
Agalya
A
Periasamy
Nadim
N
Shadiac
Amritha
A
Amalraj
Soňa
S
Garajová
Yagnesh
Y
Nagarajan
Shane
S
Waters
Haydyn D T
HD
Mertens
Maria
M
Hrmova
D060054:Agrobacterium / Q000378:metabolism; D002384:Catalysis; D002474:Cell-Free System; D004926:Escherichia coli / Q000378:metabolism; D005947:Glucose / Q000737:chemistry; D005964:Glucosyltransferases / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry*; D046529:Microscopy, Electron, Transmission / Q000379:methods; D053758:Nanoparticles / Q000737:chemistry*; D036103:Nanotechnology / Q000379:methods*; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D014176:Protein Biosynthesis; D011506:Proteins / Q000737:chemistry; D012333:RNA, Messenger / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013501:Surface-Active Agents / Q000737:chemistry; D014357:Trypsin / Q000737:chemistry; D014530:Uridine Diphosphate / Q000737:chemistry; D047071:beta-Glucans / Q000737:chemistry*
D008081:Liposomes; D010455:Peptides; D011506:Proteins; D012333:RNA, Messenger; D013501:Surface-Active Agents; D047071:beta-Glucans; D014530:Uridine Diphosphate; C038459:curdlan; D005964:Glucosyltransferases; C020570:1,3-beta-glucan synthase; D014357:Trypsin; D005947:Glucose