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2001 | 48 | 2 |

Tytuł artykułu

The cell-free protein biosynthesis - applications and analysis of the system

Warianty tytułu

Języki publikacji

EN

Abstrakty

EN
The in vitro protein biosynthesis has the potentials to be come a powerful technology for biochemical research. Beside the determination of structure and function the in vi­tro evolution of proteins is also of great interest. The system described was used to produce bo vine heart fatty acid binding protein (FABP) and bacterial chloramphenicol acetyltransferase (CAT) with and with out fu­sion of the Strep-tag II af fin ity pep tide. The proteins were purified after and during protein biosynthesis by using a StrepTactin Sepharose matrix. No significant influence of the Strep-tag and the conditions during the af fin ity chro ma tog ra phy on mat li­ra tion or activity of the protein was observed. The in vitro evolution of proteins is feasible by means of ribosome display. The selec­tion of a specific mRNA coding for a short ened FABP with a N-terminal His-tag via the accompanying protein property was shown. Goal of the selection was to bind the FABP via the His-tag on Ni(II)-IDA-agarose. Af ter nine cy cles of tran scrip tion, trans la tion, affinity selection and RT-PCR the protein with the His-tag could be enriched 108-fold. In or der to cor re late a pos si ble re la tion ship be tween changes in pro tein pop u la tion and bi o log i cal func tion stud ies were ini ti ated in which 2-dimensional pro tein pat terns of the to tal in vitro sys tem were com pared af ter 0 and 2 h re ac tion time. The very in ter- esting findings are that a number of proteins disappear, while others are newly formed dur ing protein synthesis.

Wydawca

-

Rocznik

Tom

48

Numer

2

Opis fizyczny

p.453-465,fig.

Twórcy

autor
  • Freie Universitat Berlin, Thielallee 63, D-14195 Berlin, Germany
autor
autor

Bibliografia

  • 1.Appel, R.D., Hochstrasser, D.F., Funk, M., Vargas, J.R., Pellegrini, C., Muller, A.F. & Scherrer, J.R. (1991) The MELANIE project: From a biopsy to automatic protein map interpretation by computer. Electrophoresis 12, 722-735.
  • 2.Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254.
  • 3.Cronenberger, J.H. & Erdmann, V.A. (1975). Stimulation of polypeptide polymerization by blocking of free sulphydryl groups in Escherichia coli ribosomal proteins. J. Mol. Biol. 95, 125-137.
  • 4.Hanes, J. & Plueckthun, A. (1997) In vitro selection and evolution of functional proteins by using ribosome display. Proc. Natl. Acad. Sci. U.S.A. 94, 4937-4942.
  • 5.Haukanes, B.I. & Kvam, C. (1993) Application of magnetic beads in bioassays. Biotechnology 11, 60-63.
  • 6.Heukeshoven, J. & Dernick, R. (1985) Simplified method for silver staining of proteins in polyacrylamide and the mechanism of silver staining. Electrophoresis 6, 103-112.
  • 7.Holschuh, K. & Gassen, H.G. (1982) Mechanism of translocation. Binding equilibria between the ribosome, mRNA analogues, and cognate tRNAs. J. Biol. Chem. 257, 1987-1992.
  • 8.Klose, J. & Kobalz, U. (1995) Two-dimensional electrophoresis of proteins: An updated protocol and implications for a functional analysis of the genome. Electrophoresis 16, 1034-1059.
  • 9.Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.
  • 10.Mattheakis, L.C., Bhatt, R.R. & Dower, W.J. (1994) An in vitro polysome display system for identifying ligands from very large peptide libraries. Proc. Natl. Acad. Sci. U.S.A. 91, 9022-9026.
  • 11.Merk, H., Stiege, W., Tsumoto, K., Kumagai, I. & Erdmann, V.A. (1999) Cell-free expression of two single-chain monoclonal antibodies against lysozyme: Effect of domain arrangement on the expression. J. Biochem. 125, 328-333.
  • 12.Moore, J.C. & Arnold, F.H. (1996) Directed evolution of a para-nitrobenzyl esterase for aqueous-organic solvents. Nat. Biotechnol. 14, 458-467.
  • 13.Nygren, P.-A., Stahl, S. & Uhlen, M. (1994) Engineering proteins to facilitate bioprocessing. Trends Biotechnol. 12, 184-188.
  • 14.Osborne, S.E. & Ellington, A.D. (1997) Nucleic acid selection and the challenge of combinatorial chemistry. Chem. Rev. 97, 349-370.
  • 15.Peled-Zehavi, H., Smith, C.A., Harada, K. & Frankel, A.D. (2000) Screening RNA-binding libraries by transcriptional antitermination in bacteria. Methods Enzymol. 318, 297-308.
  • 16.Roberts, R.W. & Szostak, J.W. (1997) RNA-peptide fusions for the in vitro selection of peptides and proteins. Proc. Natl. Acad. Sci. U.S.A. 94, 12297-12302.
  • 17.Ryabova, L.A., Ortlepp, S.A. & Baranov, V.I. (1989) Preparative synthesis of globin in a continuous cell-free translation system from rabbit reticulocytes. Nucleic Acids Res. 17, 4412.
  • 18.Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular cloning. Cold Spring Harbour Laboratory Press.
  • 19.Schatz, P.J., Cull, M.G., Martin, E.L. & Gates, C.M. (1996) Screening of peptide libraries linked to lac repressor. Methods Enzymol. 267, 171-191.
  • 20.Schindler, P.T., Macherhammer, F., Arnold, S., Reuss, M. & Siemann, M. (1999) Investigation of translation dynamics under cell-free protein biosynthesis conditions using high-resolution two-dimensional gel electrophoresis. Electrophoresis 20, 806-812.
  • 21.Schmidt, T.G.M., Koepke, J., Frank, R. & Skerra, A. (1996) Molecular interaction between the Strep-tag affinity peptide and its cognate target, streptavidin. J. Mol. Biol. 255, 753-766.
  • 22.Siemann, M., Schindler, P.T., Arnold, S., Baumann, S., Scharnweber, K. & Reuss, M. (2000) State of protein phosphorylation among limitations in cell-free protein biosynthesis. Eur. J. Biochem. in press.
  • 23.Smith, G.P. & Petrenko, V.A. (1997) Phage display. Chem. Rev. 97, 391-410.
  • 24.Spirin, A.S., Baranov, V.I., Ryabova, L.A., Ovodov, F.Y. & Alakhov, Y.B. (1988) A continuous cell-free translation system capable of producing polypeptides in high yield. Science 242, 1162-1164.
  • 25.Stiege, W. & Erdmann, V.A. (1995) The potentials of the in vitro protein biosynthesis system. J. Biotechnol. 41, 81-90.
  • 26.Triana-Alonso, F.J., Dabrowski, M., Wadzack, J. & Nierhaus, K.H. (1995) Self-coded 3'-extension of run-off transcripts produces aberrant products during in vitro transcription with T7 RNA polymerase. J. Biol. Chem. 270, 6298-6307.
  • 27.Voss, S. & 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.
  • 28.Zhang, J.-H., Dawes, G. & Stemmer, W.P.C. (1997) Directed evolution of a fucosidase from a galactosidase by DNA shuffling and screening. Proc. Natl. Acad. Sci. U.S.A. 94, 4504- 4509. *Pres ented at theIn ter na tional Con ference on "Mo lec u larAr chi tec ture ofEvo lu tion, Pri mary and Sec­ondary Determinants" Poznan, Poland, 0ctober29-31,2000.

Typ dokumentu

Bibliografia

Identyfikatory

Identyfikator YADDA

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