Overexpression and Periplasmic Transport of 5-Enolpyruvylshikimate 3-Phosphate Synthase in E. coli

대장균에서 5-Enolpyruvylshikimate 3-Phosphate Synthase의 대량 발현 및 Periplasmic Space로의 Transport

  • 김남일 (충북대학교 자연과학대학 생명과학부) ;
  • 임재윤 (충북대학교 자연과학대학 생명과학부) ;
  • 조태주 (충북대학교 자연과학대학 생명과학부)
  • Published : 1997.03.01


5-Enolpyruvylshikimate 3-phosphate(EPSP) synthase is the sixth enzyme of the shikimate pathway that synthesizes aromatic amino acids. The enzyme is a primary target for the glyphos'lte which is a broad-spectrum and environmetally safe herbicide. As a first step toward development of glyphpsate-resistant EPSP synthase, the EPSP synthase gene(aroA) was amplified by polymerase chain reaction and cluned into pET-25b vector. In this construct. designated pET-aro, the aroA gene is expressed under control of strong T7 promoter. and the EPSP synthase is produced as a fusion protein with pelB leader at N-terminus and HSV-tag and His-tag at C-terminus. When the pET-aro clone was induced to produce the enzyme, it was found that the EPSP synthase was successfully exported to peri plasmic space. The periplasmic transport was greatly dependent on the induction temperatures. Among the induction temperatures examined($25^{\circ}C$, $30^{\circ}C$, $34^{\circ}C$ and $37^{\circ}C$). induction at $34^{\circ}C$ gave rise to maximal periplasmic transport. The recomhinant EPSP synthase could have been purified hy $Ni^{2+}$ -affinity chromatography using the His-tag. and detected hy anti-HSV -tag antibody. The recombinant EPSP synthase also hound to phosphocellulose resin and was eluted hy shikimate 3-phosphate and phosphoenolpyruvate. as expected. The recombinant EPSP synthase purified from phosphocellulose resin showed typical EPSP synthase activity.


  1. Curr. Opinion Struct Biol v.3 Phage display fo peptides and protein domains Hoess, R. H.
  2. Ann. Rev. Biochem. v.57 Amino acid biosyn-thesis inhibitors as herbicides Kishore, G. M.;D. M. Shah.
  3. Pisum sativum L. Planta v.160 Purification and properties of 5-enolpyruvylshikimate-3-phosphate synthase from seedlings Mousdale, D.M;J.R.Coggins
  4. FEBS LETT. v.154 Kinctics of 5-enolpyruvylshikimate-3-phosphate synthase inhibition by glyphosate Boocock, M. R.;J.R. Coggins
  5. Esherichia col. Biochem. J. v.213 Purification of 5-enolpyruyl shikimate-3-phosphate synthase Lewendon, A.;J. R. Coggins
  6. Curr. Opinion Struct. Biol. v.4 Phage display structure assembly and engineering of filamentous bacteriophage M13 Makowki, L.
  7. Arch. Biochem. Biophys. v.244 Overproduction of 5-enolpyruvyl-shikimate-3-phosphate syntase in a glyphosate-tolerant Petunia hybida cell line Steinrucken, H.C.;A. Schultz;N. Amrhein;C.A.Porter;R.T. Fraley
  8. Vet. Hum. Tox-iol v.34 The biological ac-tivity of glyphosate to plants and animals Smith, E.A.;F.W. Oehme
  9. Nature v.348 Phage antibodies: Filamentous phage displaying antibody variable domains McCafferty, J.;A.D. Griffiths;G. Winter;D.J. Chis-well
  10. Escherichia coli. Method Enzymol v.142 3-Phos-phoshikimate 1-carboxyvinyl transferase Lewendon, A.;J.R. Coggins
  11. Anal. Biochem. v.100 An improved assay for nanomole amounts o finorganic phosphate Lanzetta, P.A.;L.J. Alvarez;P.S. Reinach;O.A. Candia
  12. Eur. J. Biochem. v.143 5-enolpyruvyl-shikimate-3-phosphate synthase of klebsiella pneumoniae:2.Inhibition by glyphosate 〔N-phosphonomethylglycine〕 Steinrucken, H.C.;N. Armhein
  13. Ann. Rev. Biochem. v.60 The enzymology of protein trasnlocation across the Escher-ichia coli plasma membrance Wickner, W.;A.J.M. Driessen;F.-U. Hartl
  14. Ann. Rev. Biochem. v.60 The enzymology of protein translocation across the Escherichia coli plasma membrane Wickner, W.;A.J.M. Driessen;F.-U. Hartl.
  15. Bicchem. Biophys. Res. Commun. v.94 The herbicide glyphosate is a potent inhibitor of 5-enolpyruvylshikimate-3-phosphate synthase Steinrucken, H.C.;N. Armhein
  16. New Zeal Med. J. v.105 Glyphosate her-bicide poisoning experience in New Zealand Temple, W.A.;N.A. Smith
  17. J. Bilo. Chem. v.246 On the mechanism of 5-enolpyruvylshikimate-3-phosphate synthase Bondinell, W.E.;J. Vnek;P.R. Knowles;M. Specher;D.B. Sprinson
  18. Mu-tation Research v.300 Genotoxicity testing of the herbicide Round-up and its active ingredient glyphosate isopropylamine us-ing mouse bone marrow micronucleus test, Salmonella mu-tagenecity test, and Allium anaphase-telophase test Rank, J.;A.-G. Jesen;B. Skov;L.H. Pedersen;K. Jensen
  19. Biochem. J. v.246 The pentafuctional arom enzyme of Sacchromyces cerevisiac is a mosaic of monofunctional domains Duncan, K.;M.R. Edwards;J.R. Coggins
  20. Proc. Natl. Acad. Sci. v.3 Fusions of secreted proteins to alkaline phosphatase: an approach for studding protein secretion Hoffman, C. S.;A. Wright.
  21. Oxford Surveys of Plant Mol. Cell Biol. v.7 Shik-imate pathway: Why does it mean so much to so many? Sing, B.K.;D.L. Siehl;J.A. Connely
  22. Plant Physiol v.76 Selection and characterization of a carrot cell line tolerant to glyphosate Nafziger, E.D.;J.M. Widholm;H.C. Steinruecken;J. L. Kilmer
  23. Biotecchnology v.7 Production of soluble recombinant pro-teins in bacteria Schein, C. H.
  24. Methods Enzymol v.185 Engineering Escher-ichia coli to secrete heterologous gene products Stader, J.A.;T.J. Silhavy
  25. Proteins v.8 Hormone phage:an enrichment method for variant proteins with alt-ered binding properties Bass, S.;R. Greene;J.A. Wells