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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Co-translational insertion into pre-formed nanodiscs makes it possible to study cell-free synthesized membrane proteins in defined lipid environments without contact with detergents. This protocol describes the preparation of essential system components and the critical parameters for improving expression efficiency and sample quality.

Abstract

Cell-free expression systems allow the tailored design of reaction environments to support the functional folding of even complex proteins such as membrane proteins. The experimental procedures for the co-translational insertion and folding of membrane proteins into preformed and defined membranes supplied as nanodiscs are demonstrated. The protocol is completely detergent-free and can generate milligrams of purified samples within one day. The resulting membrane protein/nanodisc samples can be used for a variety of functional studies and structural applications such as crystallization, nuclear magnetic resonance, or electron microscopy. The preparation of basic key components such as cell-free lysates, nanodiscs with designed membranes, critical stock solutions as well as the assembly of two-compartment cell-free expression reactions is described. Since folding requirements of membrane proteins can be highly diverse, a major focus of this protocol is the modulation of parameters and reaction steps important for sample quality such as critical basic reaction compounds, membrane composition of nanodiscs, redox and chaperone environment, or DNA template design. The whole process is demonstrated with the synthesis of proteorhodopsin and a G-protein coupled receptor.

Introduction

Membrane proteins (MPs) are challenging targets in protein production studies due to their insolubility in aqueous environments. Conventional MP production platforms comprise cell-based systems such as E. coli, yeast, or eukaryotic cells. The synthesized recombinant MPs are either extracted from cell membranes or refolded from inclusion bodies1. After detergent solubilization, MPs can be transferred into suitable membrane environments by established in vitro reconstitution protocols. Besides vesicles and liposomes, MP reconstitution into planar membranes in the form of nanodiscs2 or salipro3

Protocol

1. Preparation of S30 lysate

  1. Day 1: Streak out cells from glycerol stocks on an LB agar plate and incubate at 37 °C overnight.
  2. Day 2: Inoculate 200 mL of LB medium with the cells from the agar plate and incubate at 37 °C for 12-14 h.
  3. Day 3: Inoculate 10 L of sterile YPTG medium (10 g/L yeast extract, 16 g/L tryptone, 5 g/L NaCl, 19.8 g/L glucose, 4.4 mM KH2PO4, 8 mM K2HPO4) tempered to .......

Representative Results

The impact of fine-tuning reaction compounds on the final yield or quality of synthesized MPs is exemplified. A frequent routine approach is to adjust the optimal Mg2+ concentration in CF reactions by expression of green fluorescent protein (GFP) as a convenient monitor of system efficiency. As an example, GFP was synthesized from a pET-21a(+) vector at Mg2+ concentrations between 14 and 24 mM (Figure 2). SDS-PAGE analysis identified the optimal Mg2+ concentr.......

Discussion

The setup and strategies to optimize the CF expression and co-translational insertion of functional MPs into nanodiscs are described. The required equipment comprises a bioreactor, a French press device or similar, an UV/VIS and fluorescence reader, CF reaction vessels suitable for a two-compartment configuration setup, and a temperature-controlled incubator. Further standard equipment are centrifuges for harvesting E. coli cells as well as tabletop centrifuges reaching at least 30,000 x g for preparati.......

Acknowledgements

We would like to thank the Deutsche Forschungsgemeinschaft (DFG) grant BE1911/8-1, the LOEWE project GLUE, and the collaborative research center Transport and Communication across Membranes (SFB807) for financial support.

....

Materials

NameCompanyCatalog NumberComments
1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (DMPG)Avanti Polar Lipids (USA)840445P
1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)Avanti Polar Lipids (USA)850345C
1,2-dioleoyl-sn-glycero-3-phosphocholine (sodium salt) (DOPC)Avanti Polar Lipids (USA)850375C
1,2 dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) (sodium salt) (DOPG)Avanti Polar Lipids (USA)840475C
1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC)Avanti Polar Lipids (USA)850457C
1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (POPG)Avanti Polar Lipids (USA)840034C
2-Amino-2-(hydroxymethyl)-propan-1,3-diol (Tris)Carl Roth (Germany)4855
2-MercaptoethanolCarl Roth (Germany)4227
2-PropanolCarl Roth (Germany)9781
[3H]-dihydroalprenolol HydrochlorideAmerican Radiolabeled Chemicals (USA)ART0134
Acetyl phosphate lithium potassium salt (ACP)Merck (Germany)1409
Adenosine 5’-triphosphate (ATP)Sigma Aldrich (Germany)A9251
Alprenolol hydrochlorideMerck (Germany)A0360000
Anion exchange chromatography column material: Q-sepharose®Sigma-Aldrich (Germany)Q1126
Autoclave Type GE 446EC-1Gettinge (Germany)
Bioreactor Type 884 124/1B.Braun (Germany)
CentrifugeSorvall RC12BP+; Thermo Scientific (Germany); Sorvall RC-5C; Thermo Scientific (Germany); Mikro 22 R; Hettich (Germany)
Cholic acidCarl Roth (Germany)8137
Coomassie Brilliant Blue R250Carl Roth (Germany)3862
Culture flasks 500 ml baffled flasks, 2 l baffled flasksSchott Duran (Germany)
Cytidine 5'-triphosphate disodium saltSigma-Aldrich (Germany)C1506
D-glucose monohydrateCarl Roth (Germany)6780
Di-potassiumhydrogen phosphate trihydrateCarl Roth (Germany)6878
Dialysis tubing SpectrumTM Labs Spectra/PorTM 12-14 kD MWCO Standard RC tubingFisher Scientific (Germany)8700152
DithiothreitCarl Roth (Germany)6908
EthanolCarl Roth (Germany)K928
Folinic acid calcium salt hydrateSigma-Aldrich (Germany)47612
French pressure cell disruptorSLM; Amico Instruments (USA)
GlycerolCarl Roth (Germany)3783
Guanosine 5'-triphosphate di-sodium salt (GTP)Sigma-Aldrich (Germany)G8877
Hydrochloric AcidCarl Roth (Germany)K025
IMAC column: HiTrap IMAC HP 5 mLGE Life Sciences (Germany)GE17-5248
ImidazoleCarl Roth (Germany)3899
Isopropyl-β-D-thiogalactopyranosid (IPTG)Carl Roth (Germany)2316
KanamycinCarl Roth (Germany)T832
L-AlanineCarl Roth (Germany)3076.1
L-ArginineCarl Roth (Germany)6908
L-AsparagineCarl Roth (Germany)HN23
L-Aspartic AcidCarl Roth (Germany)T202
L-CysteineCarl Roth (Germany)T203
L-Glutamic AcidCarl Roth (Germany)3774
L-GlutamineCarl Roth (Germany)3772
L-GlycineCarl Roth (Germany)3187
L-HistidineCarl Roth (Germany)3852
L-IsoleucineCarl Roth (Germany)3922
L-LeucineCarl Roth (Germany)1699
L-LysineCarl Roth (Germany)4207
L-MethionineCarl Roth (Germany)9359
L-ProlineCarl Roth (Germany)1713
L-PhenylalanineCarl Roth (Germany)1709
L-SerineCarl Roth (Germany)4682
L-ThreonineCarl Roth (Germany)1738
L-TryptophaneCarl Roth (Germany)7700
L-TyrosineCarl Roth (Germany)T207
MD100 dialysis unitsScienova (Germany)40077
N-2-Hydroxyethylpiperazine-N'-2-ethansulfonic acid (HEPES)Carl Roth (Germany)6763
n-dodecylphosphocholineAntrace (USA)F308S
PAGE chamber: Mini-Protean Tetra CellBiorad (Germany)
PAGE gel casting system: Mini Protean Handcast systemsBiorad (Germany)
PAGE gel power supply: Power Pac 3000Biorad (Germany)
Tryptone/peptone from caseineCarl Roth (Germany)6681
Peristaltic pump: ip-12Ismatec (Germany)
Phosphoenol pyruvate monopotassium saltSigma Aldrich (Germany)860077
Potassium dihydrogen phosphateCarl Roth (Germany)P018
Potassium acetateCarl Roth (Germany)4986
Potassium chlorideCarl Roth (Germany)6781
Pyruvate KinaseRoche (Germany)10109045001
Scintillation counter: Hidex 300 SLHidex (Finland)
SDS pelletsCarl Roth (Germany)8029
Sodium azideSigma-Aldrich (Germany)K305
Sodium chlorideCarl Roth (Germany)P029
Spectrophotometer NanodropPeqlab (Germany)
Spectrophotometer/fluorescence reader Spark®Tecan (Switzerland)
tRNA (E. coli)Roche (Germany)10109550001
Ultra sonificatorLabsonic U, B. Braun (Germany)
Uridine 5’-triphosphate tri-sodium salt (UTP)Sigma-Aldrich (Germany)U6625
Y-30 antifoamSigma-Aldrich (Germany)A6457
Yeast extractCarl Roth (Germany)2904

References

  1. Pandey, A., Shin, K., Patterson, R. E., Liu, X. Q., Rainey, J. K. Current strategies for protein production and purification enabling membrane protein structural biology. Biochemistry and Cell Biology. 94 (6), 507-527 (2016).
  2. Ritchie, T. K., et al.

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