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

Abstract

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.

Single-channel electrophysiology of cell-free expressed ion channels by direct incorporation in lipid bilayers

“…[12][13][14] Interestingly, when liposomes are added to the cell-free reaction mixture, spontaneous reconstitution has been demonstrated for a variety of cell-free expressed membrane proteins, including stearyl-CoA desaturase, glucan synthase, ATP synthase, DesK thermosensor, endothelin receptors A and B, bacteriorhodopsin, connexin-43, aquaporin Z, and the ion channels Kcv and KcsA. [15][16][17][18][19][20][21][22][23][24] Given that incorporation of protein into the liposome cannot be facilitated by translocon components as these are not present in the lysate, it has been postulated that the presence of detergents, trace amounts of native lipids, or a close ribosome-liposome proximity aids protein insertion in the lipid bilayer of the liposomes. [12][13][14] In principle, the bilayer self-insertion of cell-free expressed membrane proteins can be exploited as a purication method for ion channel electrophysiology, as demonstrated in a small number of recent studies.…”

A Barley Efflux Transporter Operates in a Na+-Dependent Manner, as Revealed by a Multidisciplinary Platform

“…The sources of oligonucleotide primers, restriction and DNA modifying enzymes, plasmid extraction kits, EDTA-free Complete Protease Inhibitor Cocktail tablets, and other chemicals were described previously (Periasamy et al, 2013). Benzonase (40 units/mL) was from Invitrogen.…”

“…A mixture of POPC:POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine):POPG [1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol)]:cholesterol = 6:3:1:0.1 (by weight) was used at 10 mg/mL. For synthesis of Bot1 with surfactants, liposomes were replaced by Brij-58, n-dodecyl-b-D-maltoside, n-decyl-b-D-maltoside, digitonin, Triton X-100, Tween 80, or acetylated (Ac) surfactant peptides Ac-AAAAAAD (Ac-Ala-Ala-Ala-Ala-Ala-Ala-Asp), Ac-IIID (Ac-Ile-Ile-Ile-Asp), and Ac-LLLK-NH 2 (Ac-Leu-Leu-Leu-Lys-NH 2 ) in the translation reaction as described (Periasamy et al, 2013). Samples from WG-CFPS were evaluated by immuno-dot-blot analyses, and the positive samples were analyzed by SDS-PAGE coupled with immunoblot analyses using either a mouse IgG2a isotype anti-6xHis monoclonal antibody (Clontech) or a crude serum raised through immunization of rabbits with the CSVDKDLKSLKDAVLREGDE peptide (residues 419 to 437, positioned between membrane a-helices nine and ten) derived from Bot1.…”

“…Wheat germ cell-free protein synthesis (WG-CFPS) was used to produce full-length barley Bot1 via cotranslational insertion into liposomes in a decoupled bilayer mode (Periasamy et al, 2013). The translation reaction was performed in the presence of a variety of liposomes (Figure 1; Supplemental Figure 1), including a mixture of lipids to mimic the composition of a plant membrane (Magdy et al, 1994).…”

“…To conduct WG-CFPS of Bot1 in the presence of unilamellar liposomes and surfactants, the synthesis was conducted as described previously (Periasamy et al, 2013), through uncoupled transcription and translation at 25°C for 24 h in a bilayer mode. DMPC, POPC (1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine), and asolectin lipids were prepared at 50 mg/mL.…”