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Hands-on Demonstration of Wheat Germ Cell-free Translation

Hands-on Demonstration of Wheat Germ Cell-free Translation. Shin-ichi Makino, Michael A. Goren, Yuko Matsubara, Kazuyuki Takai, Hidenori Hayashi, Brian G. Fox, John L. Markley and Yaeta Endo Center for Eukaryotic Structural Genomics University of Wisconsin-Madison, Madison WI USA

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Hands-on Demonstration of Wheat Germ Cell-free Translation

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  1. Hands-on Demonstration of Wheat GermCell-free Translation Shin-ichi Makino, Michael A. Goren, Yuko Matsubara, Kazuyuki Takai, Hidenori Hayashi, Brian G. Fox, John L. Markley and Yaeta Endo Center for Eukaryotic Structural Genomics University of Wisconsin-Madison, Madison WI USA Cell-Free Science and Technology Research Center Ehime University, Matsuyama Japan

  2. Your cell-free translation kit

  3. Steps to assemble your translation reaction

  4. Flowchart for CESG structural studies

  5. Notes • All glassware must be baked for 3 h at 180˚C to eliminate contaminating RNase. • In order to prevent RNase contamination from hands or saliva, wear gloves and keep the mouth closed while handling reagents. • All the buffers must be sterilized by passage through a 0.2 μm filter, and stored at -20˚C unless otherwise indicated. • Plasmid DNA prepared by commercially available kits often contains a contamination of RNase. This contaminant must be removed for successful transcription and translation. • Contaminating wheat germ extract bands in the first IMAC purification step can be diminished by use of WEPRO2240H wheat germ extract, which has been pretreated with an IMAC resin to remove these endogenous contaminants.

  6. Primer design • Use a two-step PCR to create needed gene functionality • 1st PCR primers are gene specific; 2nd PCR primers are universal • Match the 5’ of gene starting with 3rd codon of the ORF; append the TEV protease site and SgfI site in order to liberate Ser as N-terminus • Match 3’ of gene with stop codon; append PmeI site

  7. Plasmid • SP6 promoter • TMV Omega sequence • sacB-CAT toxic selection • pF1K homology region • CESG plasmids are available in the PSI-MR • Use Flexi Vector cloning • SEQUENCE-VERIFY YOUR GENE!!!!

  8. Plasmid preparation Steps Commentary Plasmid DNA is a valuable reagent for the transcription reaction You need more quantity and higher purity than for cell-based cloning Ethanol and and precipitants are deleterious • Transformed cells grown in 2YT • Marligen high purity maxi-prep kit • Proteinase K treatment of partially purified plasmid to remove all residual RNAse activity • Phenol:chloroform extraction to remove proteinase K • Acetate/ethanol precipitation • Carefully dry the DNA pellet • Re-suspend in 18 MΩ water; use A260 measurements to prepare 1 µg/µL

  9. Reagents for transcription • Transcription Buffer (TB+Mg, 5) is 400 mM HEPES-KOH, pH 7.8, containing 100 mM magnesium acetate, 10 mM spermidine hydrochloride, and 50 mM DTT. Store this buffer at -20˚C. • An NTP solution containing 25 mM each of ATP, GTP, CTP, and UTP is prepared from 0.2 μm filter-sterilized, 100 mM solutions of each NTP prepared in Milli-Q water. NTP solutions are stored at -80˚C. • SP6 RNA polymerase and RNase inhibitor (RNasin) are from Promega. • The transcription mixture (TB, 2) contains 2 TB+Mg, 8 mM NTPs, 3.2 unit/μL of SP6 RNA polymerase, and 1.6 unit/μL of RNasin. This solution is prepared immediately before use.

  10. Transcription reaction Steps Commentary If the transcription reaction is proceeding correctly, a white precipitate of magnesium pyrophosphate will form and make the transcription solution turbid. In order to avoid co-precipitation of mRNA, the reaction should not be chilled. • In a 96-well PCR plate, dispense 2.5 µL of the transcription mixture into each well and add 2.5 μL of each separate plasmid DNA prepared as before to separate wells of the PCR plate. • Tightly cap the wells to avoid concentration of the samples by evaporation. Incubate the transcription reaction at 37˚C for 4 h. • This RNA solution is used in translation reactions without further purification.

  11. Wheat germ extract and proteins • The wheat germ cell-free extract is WEPRO2240 from CellFree Sciences, Ltd. (Yokohama, Japan). This preparation has 240 OD260 per mL and is prepared without amino acids. Store the extract at -80˚C. • Contaminating wheat germ extract bands in the first IMAC purification step can be diminished by use of WEPRO2240H wheat germ extract, which has been pretreated with an IMAC resin to remove these endogenous contaminants. • Creatine kinase is from Roche Applied Sciences (Indianapolis, IN). Dissolve in Milli-Q water to make 50 mg/mL and store at -80˚C. Dilute the stock solution to 1 mg/mL prior to use.

  12. Dialysis buffer (substrates) • Unlabeled amino acids are from Advanced ChemTech (Louisville, KY) • 15N and 13C,15N-labeled amino acids are from Cambridge Isotope Laboratories (Andover, MA). • A mixture of the 20 unlabeled amino acids is prepared in Milli-Q water with each amino acid present at 2 mM. The 15N-labeled amino acids are prepared at 8 mM, and the 13C,15N-labeled amino acids are prepared at 5 mM, also in Milli-Q water. Do not filter these preparations because some amino acids are not dissolved in these solutions. • Dialysis Buffer (DB, 5) is prepared from 120 mM HEPES-KOH, pH 7.8, and contains 500 mM potassium acetate, 12.5 mM magnesium acetate, 2 mM spermidine hydrochloride, 20 mM DTT, 6 mM ATP, 1.25 mM GTP, 80 mM creatine phosphate, and 0.025% (w/v) sodium azide. Store this buffer at -80˚C. • An aliquot of 1 DB containing amino acids is prepared by dilution of 5 DB with Milli-Q water and addition of the appropriate amino acid mixture to 0.3 mM. Sonicate the mixture for 5 min, and then pass the solution through a 0.2 µm filter.

  13. Cofactors, metals, other additives • Metal content is shown at right; may require supplementation • Cofactors are depleted relative to level of protein translation • Not much lipid present • Detergents can be added; we prefer using liposomes

  14. Small-scale translation reaction • Prepare the translation mixture. Each individual translation mixture consists of 6.25 μL of water, 2.75 μL of 5 DB, 3.75 μL of 2 mM unlabeled amino acids, 1 µL of 1 mg/mL creatine kinase, and 6.25 μL of WEPRO2240 wheat germ extract. • Depending on the number of separate reactions to be performed, scale these volumes and include ~10% extra volume in order to fill each translation well and account for handling losses. • Add 125 μL of 1 DB with amino acids to a well of a microtitre plate. Mix 20 μL of translation mixture with 5 µL of transcription reaction to make 25 μL reaction. Execute a bi-layer method translation by injecting the 25 µL reaction under the 1 DB with amino acids. Continue the reaction for 20 h at 26˚C without disturbing the bi-layer.

  15. Translation screening—SDS PAGE SDS-PAGE analysis of proteins (A-H) soluble (S) and insoluble (P) fractions of expressed proteins. The expressed proteins are marked with an arrowhead. The amount of total protein expressed is estimated by visual inspection and comparison to the intensity of the 31 kDa marker band in the molecular weight standards, which is present at 0.15 µg. An H total expression rating corresponds to greater than 2.5 µg of protein expressed per translation reaction; an M total expression rating corresponds to more than 1.25 µg but less than 2.5 µg of protein expressed per translation reaction; a W total expression rating corresponds to less than 1.25 µg of protein expressed per translation reaction, but still detectable; and a U rating corresponds to an uncertain determination, possibly because of no detectable expression or overlap with endogenous wheat germ protein bands. The solubility is assessed by comparison of the intensity of the corresponding protein bands in the soluble and insoluble translation products gels. A high solubility rating (H) is assigned when the soluble translation band has 3 or greater of stained intensity than the insoluble translation band. A medium solubility rating (M) is assigned when the soluble translation band has approximately equal stained intensity with the insoluble translation band. A weak solubility rating (W) is assigned when the soluble translation band has less stained intensity than the insoluble translation band, and an uncertain solubility rating (U) corresponds to an uncertain determination, possibly because of no detectable expression or because of overlap with endogenous wheat germ protein bands. The proteins investigated, identified by their Gene Ontology numbers, and their purification ratings are as follows: A, GO.74329, expression rating H, solubility rating H; B, GO.34351, expression rating H, solubility rating W; C, GO.70653, expression rating H, solubility rating W; D, GO.7312, expression rating H, solubility rating M; E, GO.24674, expression rating H, solubility rating H; F, GO.79368, expression rating M, solubility rating M; G, GO.37540, expression rating H, solubility rating W; H, GO.80048, expression rating H, solubility rating M.

  16. Purification screening—His tag SDS-PAGE analysis of proteins purified from the small-scale cell-free translation reaction used to screen for protein production, solubility, and likelihood of success in large-scale purification. The two proteins observed in all lanes (~50 kDa, marked with black dots) are endogenous wheat germ proteins that co-purify with His6-tagged proteins using IMAC (See Note 5). The successfully purified protein of sample A is marked with a white star to show an example. The Gene Ontology numbers are the same as in the legend of Translation Screening—SDS PAGE. The purification ratings are H, H, U, H, H, M, W, and H (from left to right). An H rating corresponds to a purified yield of greater than 2.5 μg per small-scale translation reaction; an M rating corresponds to a purified yield of 2.5 -1.25 μg per small-scale translation reaction; a W rating corresponds to a purified yield of less than 1.25 μg per small-scale translation reaction; and a U rating corresponds to no purified protein detected.

  17. Purification screening—membrane proteins • Soybean total extract (20% lecithin) is from Avanti Polar Lipids (Alabaster, AL). • The lipid rehydration buffer is 25 mM HEPES, pH 7.5, containing 100 mM NaCl. • Track-etch polycarbonate membranes, 0.4 µm and 0.1 µm, are from Nucleopore (Pleasanton, CA). • Liposomes are prepared by extrusion. • Accudenz is from Accurate Chemical and Scientific (Westbury, NY). Prepare 80% (w/v) and 35% (w/v) solutions in 25 mM HEPES, pH 7.5, containing 100 mM NaCl and 10% (w/v) glycerol. Store these solutions at room temperature.

  18. Large-scale translation reaction, part 1 • Prepare the plasmid DNA as described earlier. Plasmid DNA prepared by commercially available kits often contains a trace contamination of RNase. This contaminant must be removed for successful transcription and translation. • To prepare sufficient mRNA for a large-scale protein production, carry out a transcription reaction in a 50 mL conical tube with a total volume of 4 mL of 1 TB+Mg containing 4 mM NTPs, 0.05 mg/mL of plasmid DNA, 0.5 unit/μL of SP6 RNA polymerase, and 0.25 unit/μL of RNasin. Incubate the reaction at 37˚C for 3 to 5 h. If the transcription reaction is proceeding correctly, a white precipitate of magnesium pyrophosphate will form and make the transcription solution turbid. • Remove the white precipitate from the transcription reaction by centrifugation in the C0650 rotor and Allegra X-22R centrifuge for 5 min at 6230 rpm (4000  g) and 26˚C. Transfer the supernatant to a new tube. This clarified solution is used as the mRNA solution. In order to avoid co-precipitation of mRNA, the reaction should not be chilled.

  19. Large-scale translation reaction, part 2 • Add 1452 μL of 1 DB with amino acids, 48 μL of creatine kinase (50 mg/mL in water), 1000 μL of WEPRO2240, and 1500 μL of mRNA solution to an Amicon Ultra-15 (10 K MWCO) concentrator. Spin the concentrator in the C0650 rotor and Allegra X-22R centrifuge for 8 min at 5395 rpm (3000  g) and 26˚C. Add 2 mL of 1 DB with amino acids, mix gently by pipetting, and spin for 5 min. Add 1 mL of 1 DB with amino acids, mix gently by pipetting, and spin for 8 min. If the volume is less than 4 mL, add 1 DB with amino acids to achieve a reaction volume of 4 mL. • Prepare an exchanging buffer by mixing 50 mL of 1 DB with amino acids and 2.5 mL of mRNA solution. Add sufficient buffer (typically 2.5 mL) to bring the reaction volume to 6.5 mL. Centrifuge the concentrator tube as above to decrease the volume to ~4 mL, and incubate at 26˚C for 1 h. Add fresh buffer to bring the volume back to 6.5 mL and repeat the centrifugation step. This cycle is repeated 18 times. • The final yield of soluble protein from the large-scale preparation typically ranges from 0.2 to 0.7 mg of purified protein per 4 mL reaction for a protein rated H in the small-scale purification trial.

  20. Robots in use at CESG • A skilled operator can carry out all of the reactions described above by hand and achieve excellent results. The following robots are used at CESG in more extensive cell-free translation studies • GeneDecoder1000™ • Fully automated transcription and translation at 10˚C to 40˚C. Capable of yielding ~10 µg of protein in each of the 384 (4 x 96) wells in 24 h, with a 4 h transcription step and 20 h translation cycle. A typical reaction volume is 25 µL. • Protemist100™ • Large-scale synthesis robot with fully automated transcription and translation. Capable of up to 8 translation reactions in a fully automated mode over a 24-h period. In our experience, 4 mL reactions yield an average of 0.5 mg protein. • DT-II™ • Bench-top robot capable of fully automated screening or production. Screening mode carries out 24 x 1.2 mL reactions for 24 h. Production mode carries out 6 x 6 mL reactions with yield of ~1 mg per run. DT-II will perform automated purification of His- or GST-tagged proteins, including treatment with either PreScission or TEV protease to remove tags as part of the automation cycle.

  21. Summary • Wheat germ cell-free translation gives rapid access to proteins for functional and structural studies • Care with reagents is needed to assure success • Scale-up of the production of proteins identified by small-scale screening is proportional to the volume increase • Open system provides options for addition of metal and cofactors, and co-translation to produce multi-protein complexes • Membrane proteins are translated with liposomes in a functional state and easily purified

  22. Acknowledgements • This work was supported by NIGMS Protein Structure Initiative grant 1U54 GM074901 (J.L. Markley, PI, G.N. Phillips and B.G. Fox, Co-Investigators, which supports CESG), NIGMS grant P41 RR02301 (J.L. Markley, PI, which supports the National Magnetic Resonance Facility at Madison, where NMR spectroscopy was carried out), and NIGMS 50853 (B.G. Fox, PI). M.A. Goren was recipient of an NSF East Asia and Pacific Summer Institutes Fellowship. Dr. Dmitriy A. Vinarov led initial work at CESG on the development of the wheat germ cell-free system for structural genomics efforts with assistance from Ms. Ejan Tyler and Dr. Carrie Loushin Newman. • The authors thank Prof. Yaeta Endo and others at Ehime University for advice and encouragement and also the staff members of CellFree Sciences Ltd. (Yokohama, Japan) who made this approach commercially viable.

  23. References • FlexiVector Cloning • Blommel, P. G., P. A. Martin, R. L. Wrobel, E. Steffen, and B. G. Fox. 2006. High efficiency single step production of expression plasmids from cDNA clones using the Flexi Vector cloning system. Protein Expr Purif 47:562-70. • P. G. Blommel, P. A. Martin, K. D. Seder, R. L. Wrobel and B. G. Fox. (2007). Flexi Vector cloning in high throughput protein expression and purification. In Methods in Molecular Biology (S. Doyle, Ed.), The Humana Press Inc., Totowa, N.J. • Cell-free translation—screening • Tyler, R. C., D. J. Aceti, C. A. Bingman, C. C. Cornilescu, B. G. Fox, R. O. Frederick, W. B. Jeon, M. S. Lee, C. S. Newman, F. C. Peterson, G. N. Phillips, Jr., M. N. Shahan, S. Singh, J. Song, H. K. Sreenath, E. M. Tyler, E. L. Ulrich, D. A. Vinarov, F. C. Vojtik, B. F. Volkman, R. L. Wrobel, Q. Zhao, and J. L. Markley. 2005. Comparison of cell-based and cell-free protocols for producing target proteins from the Arabidopsis thaliana genome for structural studies. Proteins 59:633-43. • Vinarov, D. A., B. L. Lytle, F. C. Peterson, E. M. Tyler, B. F. Volkman, and J. L. Markley. 2004. Cell-free protein production and labeling protocol for NMR-based structural proteomics. Nat Methods 1:149-53. • Makino, S., M.A. Goren, B.G. Fox, and J.L. Markley (2009) Cell-free protein synthesis technology in NMR high-throughput structure determination. Draft provided for instructional purposes. • Cell-free translation—production for structural studies • Vinarov, D. A., C. L. Newman, E. M. Tyler, J. L. Markley, and M. N. Shahan. 2006. Wheat germ cell-free expression system for protein production. Curr Protoc Protein Sci Chapter 5:Unit 5 18. • Makino, S., M.A. Goren, B.G. Fox, and J.L. Markley (2009) Cell-free protein synthesis technology in NMR high-throughput structure determination. Draft provided for instructional purposes. • Membrane proteins, liposomes density gradient purification • Goren, M. A., and B. G. Fox. 2008. Wheat germ cell-free translation, purification, and assembly of a functional human stearoyl-CoA desaturase complex. Protein Expr Purif 62:171-78. • Makino, S., M.A. Goren, B.G. Fox, and J.L. Markley (2009) Cell-free protein synthesis technology in NMR high-throughput structure determination. Draft provided for instructional purposes.

  24. Hands-on Demonstration of Wheat GermCell-free Translation Shin-Ichi Makino, Michael A. Goren, Yuko Matsubara, Kazuyuki Takai, Hidenori Hayashi, Brian G. Fox, John L. Markley and Yaeta Endo Center for Eukaryotic Structural Genomics, University of Wisconsin-Madison, Madison WI USA and Cell-Free Science and Technology Research Center, Ehime University Ehime University, Matsuyama Japan

  25. Steps to assemble your translation reaction

  26. Time lapse photos of translation reaction

  27. SDS-PAGE analysis of translation reaction

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