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Improved Batch Translation System Based on E. coli Extract

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Cell-Free Translation Systems

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Abstract

Product removal from the continuous flow cell-free (CFCF) reactor still only partially solves some of the problems of this system, since the use of ultrafiltration membranes has some remaining limitations. These can be overcome by introducing an affinity system. A cell-free protein synthesis system has therefore been employed to produce bovine heart fatty acid binding protein (FABP) and bacterial chloramphenicol acetyltransferase (CAT) with and without fusion of the Strep-tag affinity peptide. These two fusion proteins were purified via a streptavidin and StrepTactin sepharose matrix respectively. No significant influence of the Strep-tag and the conditions during the affinity chromatogramphy on maturation or activity of the proteins were observed. In addition, quantitative removal of the fusion proteins during cell-free synthesis from a batch reaction and a semicontinuous flow cell-free (SFCF) reactor was achieved. The results document that it is possible to avoid the limitations of the ultrafiltration membranes during product removal from a CFCF reactor. The data presented show that the affinity system is also well suited for the development of a novel protein bioreactor.

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References

  1. Alimov AP, Khmelnitsky AY, Simonenko PN, Spirin AS and Chetverin AB (2000) Cell free synthesis and affinity isolation of proteins on a nanomole scale. BioTechniques 28:338–344

    Google Scholar 

  2. Baranov VI, Morozov IY, Ortlepp SA and Spirin AS (1989) Gene expression in a cell-free system on the preparative scale. Gene 84: 463–466

    Article  CAS  Google Scholar 

  3. Burks EA, Chen G, Georgiou G. and Iverson BL (1997) In vitro scanning saturation mutagenesis of an antibody binding pocket. Proc. Natl. Acad. Sci. USA 94:412–417

    Article  CAS  Google Scholar 

  4. Cornish VW and Schultz PG (1995) Site-directed mutagenisis with an expanded genetic code. Annu. Rev. Biophys. Biomol. Struct. 24: 435–462

    Article  Google Scholar 

  5. Ellman JA, Volkman BF, Mendel D, Schultz PG and Wemmer DE (1992) Site-specific isotopic labeling of proteins for NMR studies. J. Am. Chem. Soc. 114: 7959–7961

    Article  CAS  Google Scholar 

  6. Hanes J and Plückthun A (1997) In vitro selection and evolution of functional proteins by using ribosomal display. Proc. Natl. Acad. Sci. USA 94: 4937–4942

    Article  CAS  Google Scholar 

  7. Kawarasaki Y, Kawai T, Nakano H and Yamane T (1995) A long-lived batch reaction system of cell-free protein synthesis. Anal. Biochem. 226: 320–324

    Article  CAS  Google Scholar 

  8. Kigawa T and Yokoyama S (1991) A continuous cell-free protein synthesis system for coupled transcription-translation. J. Biochem. 110: 166–168

    CAS  Google Scholar 

  9. Kigawa T, Muto Y and Yokoyama S (1995) Cell-free synthesis and amino acid-selective stable isotope labeling of proteins for NMR analysis. J. Biomol. NMR 6: 129–134

    Article  CAS  Google Scholar 

  10. Kigawa T, Yabuki T, Yoshida Y, Tsutsui M, Ito Y, Shibata T and Yokoyama S (1999) Cell-free production and stable-isotope labeling of milligram quantities of protein. FEBS Letters 442: 15–19

    Article  CAS  Google Scholar 

  11. Kim DM, Kigawa T, Choi CY and Yokoyama S (1996) A highly efficient cell-free protein synthesis system from Escherichia coli. Eur. J. Biochem. 239:881–886

    CAS  Google Scholar 

  12. Kim DM and Choi CY (1996) A semicontinuous prokaryotic coupled transcription/translation system using a dialysis membrane. Biotechnol. Prog. 12:645–649

    Google Scholar 

  13. Kim DM and Swartz JR (2000) Prolonging cell-free protein synthesis by selective reagent additions. Biotechnol. Prog. 16: 385–390

    Article  CAS  Google Scholar 

  14. Kudlicki W, Kramer G and Hardesty B (1992) High efficiency cell-free synthesis of proteins: refinement of the coupled transcription/translation system. Anal. Biochem. 206:389–393

    Google Scholar 

  15. Lanar DE and Kain KC (1994) Expression-PCR (E-PCR): overview and applications. PCR Methods Appl. 4: 92–96

    Google Scholar 

  16. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685

    Article  CAS  Google Scholar 

  17. Ludlam CFC, Sonar S, Lee CP, Coleman M, Herzfeld J, RajBhandary UL and Rothschild KJ (1995) Site-directed labeling and ATR-FTIR difference spectroscopy of bacteriorhodopsin: The peptide carbonyl group of Tyr 185 is structurally active during the bR → N transition. Biochemistry 34: 2–6

    CAS  Google Scholar 

  18. Madin K, Sawasaki T, Ogasawara T and Endo Y (2000) A highly efficient and robust cell-free protein synthesis system prepared from wheat embryos: plants apparently contain a suicide system directed at ribosomal. Proc. Natl. Acad. Sci. USA 97: 559–564

    Article  CAS  Google Scholar 

  19. Marszal E and Scouten WH (1996) Dihydrofolate reductase synthesis in the presence of immobilized methotrexate. An approach to a continuous cell-free protein synthesis system. J.Mol Recognit. 9:543–548

    Google Scholar 

  20. Martemyanov KA, Spirin AS and Gudkov AT (1997) Direct expression of PCR products in a cell-free transcription/translation system: synthesis of antibacterial peptide cecropin. FEBS Lett. 414: 268–270

    Article  CAS  Google Scholar 

  21. Mattheakis LC, Bhatt RR and Dower WJ (1994) An in vitro polysome display system for identifying ligands from very large peptide libraries. Proc. Natl Acad. Sci. USA 91: 9022–9026

    Article  CAS  Google Scholar 

  22. Merk H, Stiege W, Tsumoto K, Kumagai I and Erdmann VA (1999) Cell-free expression of two single-chain monoclonal antibodies against lysozyme: Effect of domain arrangement on the expression. J. Biochem. 125: 328–333

    CAS  Google Scholar 

  23. Merk H (2000) Steigerung der Effizienz des Escherichia coli in vitro Translationssystems durch Optimierung der Nukleinsäurekomponenten. PhD Thesis, Freie Universität Berlin

    Google Scholar 

  24. Nakano H, Shinbata T, Okumura R, Sekiguchi S, Fujishiro M and Yamane T (1999) Efficient coupled transcription/translation from PCR template by hollow-fiber membrane bioreactor. Biotechnol. Bioeng. 64: 194–199

    Article  CAS  Google Scholar 

  25. Nygren P-A, Stahl S and Uhlen M (1994) Engineering proteins to facilitate bioprocessing. Trends Biotechnol. 12: 184–188

    Article  CAS  Google Scholar 

  26. Park Y, Luo J, Schultz PG and Kirsch JF (1997) Noncoded amino acids replacement probes of the aspartate aminotransferase mechanism. Biochemistry 36: 10517–10525

    Article  CAS  Google Scholar 

  27. Patnaik R and Swartz JR (1998) E. coli-based in vitro transcription/translation: in vivo-specific synthesis rates and high yields in a batch system. BioTechniques 24: 862–868

    CAS  Google Scholar 

  28. Pavlov MY, Freistroffer DV and Ehrenberg M (1997) Synthesis of region-labelled proteins for NMR studies by in vitro translation of column-coupled mRNAs. Biochimie 79: 415–422

    Article  CAS  Google Scholar 

  29. Roberts RW and Szostak JW (1997) RNA-peptide fusions for the in vitro selection of peptides and proteins. Proc. Natl Acad. Sci. USA 94:12297–12302

    Google Scholar 

  30. Sambrook J, Fritsch EF and Maniatis T (1989) Molecular cloning: A laboratory manual, 2nd edn. Cold Spring Harbour Laboratory, Cold Spring Harbor, NY

    Google Scholar 

  31. Sassenfeld HM (1990) Engineering proteins for purification. Trends Biotechnol 8:88–93

    Article  CAS  Google Scholar 

  32. Schmidt TGM and Skerra A (1993) The random peptide library-assisted engineering of a C-terminal affinity peptide, useful for the detection and purification of a functional Ig Fv fragment. Protein Eng. 6: 109–122

    Article  CAS  Google Scholar 

  33. Schmidt TGM and Skerra A (1994) One-step affinity purification of bacterially produced proteins by means of the “Strep-tag” and immobilized recombinant core streptavidin. J. Chromatog. Sect. A. 676: 337–345

    Article  CAS  Google Scholar 

  34. Schmidt TGM, Koepke J, Frank R and Skerra A (1996) Molecular interaction between the Strep-tag affinity peptide and its cognate target, streptavidin. J. Mol Biol. 255: 753–766

    Article  CAS  Google Scholar 

  35. Shaw WV and Leslie AGW (1991) Chloramphenicol acetyltransferase. Annu. Rev. Biophys. Chem. 20: 363–386

    Article  CAS  Google Scholar 

  36. Short GF, Golovine SY and Hecht SM (1999) Effects of release factor 1 on in vitro protein translation and the elaboration of proteins containing unnatural amino acids. Biochemistry 38: 8808–8819

    Article  CAS  Google Scholar 

  37. Sonar S, Lee CP, Coleman M, Patel N, Liu X, Marti T, Khorana HG, RajBhandary UL and Rothschild KJ (1994) Site-directed isotope labeling and FTIR spectroscopy of bacteriorhodopsin. Nat. Struct. Biol. 1: 512–517

    CAS  Google Scholar 

  38. Spirin AS, Baranov VI, Ryabova LA, Ovodov SY and Alakhov YB (1988) A continuous cell- free translation system capable of producing polypeptides in high yield. Science 242:1162–1164

    Google Scholar 

  39. Stiege W, Erdmann VA (1995) The potentials of the in vitro protein biosynthesis system. J. Biotechnol 41: 81–90

    Article  CAS  Google Scholar 

  40. Switzer WM and Heneine W (1995) Rapid screening of open reading frames by protein synthesis with an in vitro transcription and translation assay. Biotechniques 18: 244–248

    CAS  Google Scholar 

  41. Van der Schueren J, Robben J, Goossens K, Heremans K and Volckaert G (1996) Identification of local carboxy-terminal hydrophobic interactions essential for folding or stability of chloramphenicol acetyltransferase. J. Mol Biol 256: 8

    Article  Google Scholar 

  42. Voss S and Skerra A (1997) Mutagenesis of a flexible loop in streptavidin leads to higher affinity for the Strep-tag II peptide and improved performance in recombinant protein purification. Protein Eng. 10: 975–982

    Article  CAS  Google Scholar 

  43. Yabuki T, Kigawa T, Dohmae N, Takio K, Terada T, Ito Y, Laue ED, Cooper JA, Kainosho M and Yokoyama S (1998) Dual amino acid-selective and site-directed stable-isotope labeling of the human c-Ha-Ras protein by cell-free synthesis. J. Biomol. NMR 11: 295–306

    Article  CAS  Google Scholar 

  44. Zubay G (1973) in vitro synthesis of protein in microbial systems. Annu. Rev. Genet. 7: 267–287

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Institut für Chemie-Biochemie, Freie Universität Berlin, Thielallee 63, D-14195, Berlin, Germany

    Volker A. Erdmann

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  1. Thorsten Lamla
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  2. Volker A. Erdmann
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Editors and Affiliations

  1. Institute for Protein Research, Russian Academy of Sciences, 142292, Pushchino, Moscow Region, Russia

    Alexander S. Spirin

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© 2002 Springer-Verlag Berlin Heidelbreg

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Lamla, T., Erdmann, V.A. (2002). Improved Batch Translation System Based on E. coli Extract. In: Spirin, A.S. (eds) Cell-Free Translation Systems. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-59379-6_2

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  • DOI: https://doi.org/10.1007/978-3-642-59379-6_2

  • Publisher Name: Springer, Berlin, Heidelberg

  • Print ISBN: 978-3-642-63956-2

  • Online ISBN: 978-3-642-59379-6

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