Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지

Construction of a New Gene-Fusion Expression Vector, pMONSTER

  • Baek, Chang-Ho (Department of Genetic Engineering, Hallym University) ;
  • Wee, Sec-Han (Department of Genetic Engineering, Hallym University)
  • Published : 2000.10.01
Download PDF
⟨ Previous Next ⟩

Abstract

The fur (ferric uptake regulation) expression vector pMON2064 was modified to produce a Fur-fusion expression vector. A kinker site, factor Xa cleavage site, and several restriction endonuclease sites were introduced to facilitate easy cloning and isolating of the fusion protein. The resulting fusion expression vector, pMONSTER, was then used to make fusion expression vector, pMONSTER, was then used to make fusion proteins with $\beta$-galactosidase and the protease of the human immunodeficiency virus type 1 (HIV-1 PR). Strain SW4020 harboring the Fur $\beta$-galactosidase fusion vector produced blue colonies on a 5-bromo-4-chloro-3-indolyl-$\beta$-D-galactoside plate and the resulting 133 kDa fusion protein reacted with an anti-Fur antibody. The strain harboring the Fur-HIV-1 PR fusion vector produced a 29 kDa fusion protein, which also reacted with an anti-Fur antibody. The Fur-HIV-1 PR fusion protein was purified by a single column application that was designed to isolate the Fur protein. The purified Fur-HIV-1 PR fusion protein digested with factor Xa cleaved a recombinant Gag protein to release smaller fragments, including a p24 capsid protein. The Fur-HIV-1 PR fusion protein itself did not exhibit any proteolytic activity.

Keywords

References

  1. J. Biol. Chem v.260 Spacingof the -10 and -35 regions in the tac promoter.Effect on its in vivo activity Brosius, J.;M. Erfle;J. Storella
  2. EMBO J. v.12 Variation of half-site organization and DNA looping by araC protein Carra, J. H.;R. F. Schleif
  3. Proc. Natl. Acad. Sci. USA v.80 The tac promoter: A Functional hybrid derived from the trp and lac promoters de Boer, H. A.;L. J. comstock;M. Vasser
  4. Biochemistry v.25 Proteolytic processing of human factor Ⅷ. Correlation of specific cleavages by thrombin, factor Xa, and activated protein C with activation and inactivation of factor Ⅷ coagulant activity Eaton, D.;H. Rodriguez;G. H. Vehar
  5. Biochemostry v.11 Bovine factor χ1 (stuart factor).Mechanism of activation by protein from Russell's viper venom Fujikawa, K.;M. E. Legaz;E. W. Davie
  6. J. Bacteriol v.177 Tight regulation, modulation, and high-level expression by vectors containning the arabinose PBAD promoter Guzman, L. M.;D. Belin;M. J. Carson;J. Beckwith
  7. Anal. Biochem v.202 New vectors for high level expression of recombinant protein in bacteria Hakes, D. J.;J. E. Dixon
  8. Invitrogen catalogue 2000 Overview of Prokaryotic Expression System
  9. Methods Enzymol v.90 Maltose-binding protein from Escherichia coli Kellermann, O. K.;T. Ferenci
  10. Embo J. v.5 Production of specific antibodies against protein A fusion proteins Lowenadler, B.;B. Nilsson;L. Abrahmsen;T. Moks;L. Ljungqvist;E. Holmgren;S. Paleus;S. Josephson;L. Philipson;M. Uhlen
  11. Nature v.343 Inhibition of HIV-1 protease in infected T-lymphocytes by synthetic peptide analogues Meek, T. D.;D. M. Lambert;G. B. Dreyer;T. J. Carr;T. A. Tomaszek Jr;M. L. Moore;J. E. Stickler;C. Debouck;L. J. Hyland;T. J. Mattews;B. W. Metcalf;S. R. Petteway
  12. J. Virol v.62 The gag gene products of human immunodeficiency virus 1: Alignment within the gag open reading frame, identification of posttranslational modifications, and evidence for alternative gag precursors Mervis, R. J.;N. Ahmad;E. P. Lillehoj;M. G. Raum;F. H. Salazar;H. W. Chan;S. Venkatesan
  13. Methods Enzymol v.153 Synthesis and sequence-specific proteolysis of hybrid proteins produced in Escherichia coli Nagai, K.;H. C. Thogersen
  14. J. Virol v.65 Overexpression of the gag-pol precursor from human immunodeficiency virus type 1 provial genomes results in efficient proteolytic processing in the absence of viron production Park, J.;C. D. Morrow
  15. Gene v.20 Constuction and analysis of in vivo activity of E. coli promoter hybrids and promoter mutant that alter the-35 to -10 spacing Russel, D. R.;G. N. Bennett
  16. Molecular Cloning:A Laboratory Manual, 2nd ed. Cold Spring Habor Lab. Press
  17. Gene v.67 Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase Smith, D. B.;K. S. Johnson
  18. J. Microbiol. Biotechnol v.9 Expression and characterization of human T-cell leukemia virus type-1 Env and Gag proteins Son, Kyung Hwa;Byung-Moon Kim;Yaik-Youl Lee;Seong-Ryong Kim;Kun-Soo Kim;Jeong Kuk Lee;Jai Myung Yang
  19. J. Mol. Biol. v.11189 Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes Studier, F. W.;B. A. Moffatt
  20. Biol. Metals v.1 Expression, isolation and properties of Fur (ferric uptake regulation) protein of Escherichia coli K 12 Wee, S.;J. B. Neilands;M. L. bittner;B. C. Hemming;B. R. Haymore;R. Seetharam