Robotic large-scale application of wheat cell-free translation to structural studies including membrane proteins.

The use of the Protemist XE, an automated discontinuous-batch protein synthesis robot, in cell-free translation is reported. The soluble Galdieria sulphuraria protein DCN1 was obtained in greater than 2mg total synthesis yield per mL of reaction mixture from the Protemist XE, and the structure was subsequently solved by X-ray crystallography using material from one 10 mL synthesis (PDB ID: 3KEV). The Protemist XE was also capable of membrane protein translation. Thus human sigma-1 receptor was translated in the presence of unilamellar liposomes and bacteriorhodopsin was translated directly into detergent micelles in the presence of all-trans-retinal. The versatility, ease of use, and compact size of the Protemist XE robot demonstrate its suitability for large-scale synthesis of many classes of proteins.

[1]  Peter Güntert,et al.  Optimal isotope labelling for NMR protein structure determinations , 2006, Nature.

[2]  Tomio Ogasawara,et al.  A bilayer cell‐free protein synthesis system for high‐throughput screening of gene products , 2002, FEBS letters.

[3]  Michael D. Abràmoff,et al.  Image processing with ImageJ , 2004 .

[4]  A. Agostiano,et al.  Light‐dependent and Biochemical Properties of Two Different Bands of Bacteriorhodopsin Isolated on Phenyl‐Sepharose CL‐4B , 1999 .

[5]  M. El-Sayed,et al.  CD spectrum of bacteriorhodopsin: Best evidence against exciton model. , 1991, Biophysical journal.

[6]  M. Jackson,et al.  The Hallucinogen N,N-Dimethyltryptamine (DMT) Is an Endogenous Sigma-1 Receptor Regulator , 2009, Science.

[7]  G. Phillips,et al.  Comparison of cell‐based and cell‐free protocols for producing target proteins from the Arabidopsis thaliana genome for structural studies , 2005, Proteins.

[8]  Kazuyuki Takai,et al.  Practical cell-free protein synthesis system using purified wheat embryos , 2010, Nature Protocols.

[9]  D. Oesterhelt,et al.  Isolation of the cell membrane of Halobacterium halobium and its fractionation into red and purple membrane. , 1974, Methods in enzymology.

[10]  V. Dötsch,et al.  Cell‐free expression as an emerging technique for the large scale production of integral membrane protein , 2006, The FEBS journal.

[11]  V. Dötsch,et al.  Preparative scale cell-free expression systems: new tools for the large scale preparation of integral membrane proteins for functional and structural studies. , 2007, Methods.

[12]  J. Bujnicki,et al.  Novel protein fold discovered in the PabI family of restriction enzymes , 2007, Nucleic acids research.

[13]  Y Endo,et al.  A highly efficient and robust cell-free protein synthesis system prepared from wheat embryos: plants apparently contain a suicide system directed at ribosomes. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[14]  Michael A. Goren,et al.  Wheat germ cell-free translation, purification, and assembly of a functional human stearoyl-CoA desaturase complex. , 2008, Protein expression and purification.

[15]  G. Privé,et al.  Detergents for the stabilization and crystallization of membrane proteins. , 2007, Methods.

[16]  John L Markley,et al.  Cell-free protein synthesis technology in NMR high-throughput structure determination. , 2010, Methods in molecular biology.

[17]  Michael A. Goren,et al.  Cell-free Synthesis and Functional Characterization of Sphingolipid Synthases from Parasitic Trypanosomatid Protozoa* , 2010, The Journal of Biological Chemistry.

[18]  Michael A. Goren,et al.  Cell-free translation of integral membrane proteins into unilamelar liposomes. , 2009, Methods in enzymology.

[19]  R. Neutze,et al.  Optimized in vitro and in vivo expression of proteorhodopsin: a seven-transmembrane proton pump. , 2008, Protein expression and purification.

[20]  T. Sulchek,et al.  Cell-free expression for nanolipoprotein particles: building a high-throughput membrane protein solubility platform. , 2009, Methods in molecular biology.

[21]  John L Markley,et al.  Wheat Germ Cell‐Free Expression System for Protein Production , 2006, Current protocols in protein science.

[22]  R. S. Kaplan,et al.  A strategy for identification and quantification of detergents frequently used in the purification of membrane proteins. , 2003, Analytical biochemistry.

[23]  J. Betton,et al.  Cell-free synthesis of a functional ion channel in the absence of a membrane and in the presence of detergent. , 2004, Biochemistry.

[24]  S. Yokoyama,et al.  Production of functional bacteriorhodopsin by an Escherichia coli cell‐free protein synthesis system supplemented with steroid detergent and lipid , 2009, Protein science : a publication of the Protein Society.

[25]  Yasuhiko Yoshida,et al.  Cell‐free production and stable‐isotope labeling of milligram quantities of proteins , 1999, FEBS letters.

[26]  Brian G Fox,et al.  A combined approach to improving large-scale production of tobacco etch virus protease. , 2007, Protein expression and purification.

[27]  M. Tyers,et al.  Dcn1 functions as a scaffold-type E3 ligase for cullin neddylation. , 2008, Molecular cell.

[28]  Heinz Rüterjans,et al.  High level cell-free expression and specific labeling of integral membrane proteins. , 2004, European journal of biochemistry.

[29]  V. Dötsch,et al.  Production of membrane proteins using cell‐free expression systems , 2008, Proteomics.

[30]  E. Larson,et al.  Visualization of proteins in acrylamide gels using ultraviolet illumination. , 2002, Analytical biochemistry.

[31]  J. Schetz,et al.  An unambiguous assay for the cloned human sigma1 receptor reveals high affinity interactions with dopamine D4 receptor selective compounds and a distinct structure-affinity relationship for butyrophenones. , 2008, European journal of pharmacology.

[32]  Teruo Hayashi,et al.  Sigma-1 Receptor Chaperones at the ER- Mitochondrion Interface Regulate Ca2+ Signaling and Cell Survival , 2007, Cell.

[33]  K. Rothschild,et al.  Cell-free synthesis, functional refolding, and spectroscopic characterization of bacteriorhodopsin, an integral membrane protein. , 1993, Biochemistry.

[34]  A. Ruoho,et al.  Purification and characterization of the guinea pig sigma-1 receptor functionally expressed in Escherichia coli. , 2007, Protein expression and purification.

[35]  Eric Steffen,et al.  High efficiency single step production of expression plasmids from cDNA clones using the Flexi Vector cloning system. , 2006, Protein expression and purification.

[36]  J. Markley,et al.  Wheat germ cell‐free platform for eukaryotic protein production , 2006, The FEBS journal.

[37]  Z. Zolnai,et al.  The Center for Eukaryotic Structural Genomics , 2009, Journal of Structural and Functional Genomics.

[38]  E. Bamberg,et al.  Functional cell-free synthesis of a seven helix membrane protein: in situ insertion of bacteriorhodopsin into liposomes. , 2007, Journal of molecular biology.