Cell-Free Methods | Back to Methods Home

 

Overview

Complementary cell-free and cell-based translation methods are currently being used to prepare proteins in the target classes of sphingolipid synthases, sigma-1 receptor, fatty acid desaturases, and pilin assembly complexes for functional studies and structure determination.

We are developing cell-free translation, which utilizes ENDEXT® technology, as a versatile method for studying membrane protein structure and function. TMPC’s experimental platform is based on establishing predictive behaviors for membrane proteins using robotic cell-free translation in small-scale (50 µL to 1.2 mL). Robotic translation has an advantage of simplicity and reproducibility in the provision of material used for further research and protocol development. When appropriate expression and solubilization conditions are identified, purification protocols are developed in small-scale, and then the work is transferred to different robots whose operational volume (1.2 mL to 20 mL) better supports structure determination or expanded biological studies.

 

Robotic Instrumentation

The unique suite of cell-free translation robots in use at TMPC were developed and manufactured by the CellFree Sciences Co., Ltd. (Yokohama, Japan). They are used for high-throughput screening (GenDecoder1000) or scale-up production (Protemist DTII) of milligram quantities of membrane protein for structural analysis. The newest robot, the Protemist XE, operates using a tangential flow dialysis platform and can synthesize up to 50 mg of membrane proteins in 48 h. These instruments, and the personnel and expertise to operate them, were assembled during PSI-1 and PSI-2 at CESG. No other freely accessible research site exists worldwide with this capability. We believe these robots will be a valuable resource for the PSI:Biology Network.

  GenDecoder1000 Protemist DTII Protemist100 Protemist XE
 
Typical Use HTP Screening Screening;
Characterization
Characterization;
Production
Production
Format 96 well; 4x96 well 24 well; 6 well 8 tubes 1 reaction
Total Reaction Volume 14.4-19.2 mL 33.6 mL; 24 mL 32 mL 10-20 mL
Reaction Time 24 h 14 to 24 h 18 h 12 to 48 h
Automated Transcription Yes Yes Yes No
Automated Translation Yes Yes Yes Yes
Automated Purification No Yes No No
Yield Per Reaction 1 to 5 µg 50 µg to 1 mg 0.5 to 10 mg 1 to 70 mg

 

Publications

Aly KA, Beebe ET, Chan CH, Goren MA, Sepúlveda C, Makino SI, Fox BG, Forest KT (2013) Cell-free production of integral membrane aspartic acid proteases reveals zinc-dependent methyltransferase activity of the Pseudomonas aeruginosa prepilin peptidase PilD. Microbiologyopen 2(1):94-104. PMID:23255525

Beebe ET, Makino S-I, Nozawa A, Matsubara Y, Frederick RO, Primm JG, Goren MA, Fox BG (2010) Robotic large-scale application of wheat cell-free translation to structural studies including membrane proteins. N Biotechnol 28(3):239-49. PMID:20637905

Goren MA, Fox BG (2008) Wheat germ cell-free translation, purification, and assembly of a functional human stearoyl-CoA desaturase complex. Protein Expr Purif 62(2):171-8. PMID:18765284

Goren MA, Nozawa A, Makino S, Wrobel RL, Fox BG (2009) Cell-free translation of integral membrane proteins into unilamelar liposomes. Methods Enzymol 463:647-73. PMID:19892197

Jarecki BW, Makino SI, Beebe ET, Fox BG, Chanda B (2012) Function of Shaker potassium channels produced by cell-free translation upon injection into Xenopus oocytes. Scientific Reports, in press. PMID:23301161

Makino S-i, Goren MA, Fox BG, Markley JL (2010) Cell-free protein synthesis technology in NMR high-throughput structure determination. Methods Mol Biol 607:127-47. PMID:20204854

Sevova ES, Goren MA, Schwartz KJ, Hsu FF, Turk J, Fox BG, Bangs JD (2010) Cell-free synthesis and functional characterization of sphingolipid synthases from parasitic trypanosomatid protozoa. J Biol Chem 285(27):20580-7. PMID:20457606

Tyler RC, Aceti DJ, Bingman CA, Cornilescu CC, Fox BG, Frederick RO, Jeon WB, Lee MS, Newman CS, Peterson FC, Phillips GN Jr, Shahan MN, Singh S, Song J, Sreenath HK, Tyler EM, Ulrich EL, Vinarov DA, Vojtik FC, Volkman BF, Wrobel RL, Zhao Q, Markley JL (2005) Comparison of cell-based and cell-free protocols for producing target proteins from Arabidopsis thaliana for structural studies. Proteins 59(3):633-43. PMID:15789406

Vinarov DA, Lytle BL, Peterson FC, Tyler EM, Volkman BF, Markley JL (2004) Cell-free protein production and labeling protocol for NMR-based structural proteomics. Nat Methods 1(2):149-53. PMID:15782178

Vinarov DA, Newman CL, Markley JL (2006) Wheat germ cell-free platform for eukaryotic protein production. FEBS J 273(18):4160-9. PMID:16930128

 

Technologies at PSI SBKB Technology Portal

Expression and purification of bacteriorhodopsin using Protemist XE with detergents. (Read at Tech Portal)

Optimization of membrane protein production in E. coli and robotic purification. (Read at Tech Portal)