Genetic Stability of the Integrated Structural Gene of Guamerin in Recombinant Pichia pastoris

  • Published : 2000.08.01

Abstract

Genetic chracterstics of the structural gene of guamerin (a novel elastase inhibitor from Korean leech), integrated into the HIS4 locus of chromosomal DNA of Pichia pastoris along with the $\alpha$-factor leader sequence, were investigated. In the selected clone from candidates, two copies of the integration cassette including the structural gene copies of the integration cassette including the structural gene of guamerin were found in the integration site of the chromosomal DNA of P.pastoris. It was demonstrated that the integrated structural gene of guamerin was stable up to about 70 generations in the relay flask culture. Then, a high-cell-density culture could be fulfilled easily by DO-stat fed-batch culture, in which the cell growth and the recombinant guamerin production reached about 250 of OD600nm and 260 mg/l, respectively. Finally, it was revealed that the DNA sequence of the integrated structural gene of guamerin in P. pastoris was maintained correctly in the end of production cells of relay flask culture and high-cell-density culture.

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References

  1. J. Chem. Technol. Biotechnol. v.67 Continuous production of thrombomodulin from Pichia pastoris fermentation Chen, Y.;J. Krol;J. Cino;D. Freedman;C. White;E. Kromives
  2. Gene v.105 Production of mouse epidermal growth factor in yeast: High-level secretion using Pichia pastoris strains containing multiple gene copies Clare, J. J.;M. A. Romanos;F. B. Rayment;J. E. Rowedder;M. A. Smith;M. M. payne;K. Sreekeishna;C. A. Henwood
  3. Pichia pastoris. Bio/Technology v.7 Continuous production of a novel lysozyme via secretion from the yeast Digan, M, E.;S. V. Lair;R. A. Brierley;R. S. Siegel;M. E. Williams;s. B. Ellis;P. A. Kellaris;S. A. Provow;W. S. Craig;G. Velicelebi;M. M. Harpold;G. P. Thill
  4. J. Biol.Chem. v.270 Isolation and characterization of guamerin, a new human leukocyte elastase inhibitor from Hirudo nipponia Jung, H, I.;S. I. Kim;K. S. Ha;C. O. Joe;K. W. kang
  5. Biopharm Testing the genetic stability of recombinant DNA cell banks Kittle, J. D.;B. J. Pimentel
  6. MS Thesis Molecular cloning, sequencing, and expression of a cDNA encoding guamerin Ko, J. K.
  7. Molecular Cloning: A Laboratory Manual, (2nd ed.) Sambrook, J.;e. F. Fritsch;T. Maniatis
  8. J. Microbiol. Biotechnol. v.3 Heterologous gene expression and secretion of the anticoagulant Hirudin in a methylotrophic yeast Hansenular polymorpha Sohn, J.-H.;M. Y. Beburov;E.-S. Choi;S.-K. Rhee
  9. J. Ind. Microbiol. v.7 High-level secretion of biologically active aprotinin from the yeast pichia pastoris Vedvick, T.;R. G. Buckholz;M. Engel;M. Urcan;J. Kinney;S. Provow;R. S. Siegel;g. P. Thill
  10. Biochem. Biophys. Res. Commun v.186 High-level expression, purification, and characterization of the Kunitz-type protease inhibitor domain of protease nexin-2/amyloid B-protein precursor Wagner, S. L.;R. S. siegel;t. S. Vedvick;w. C. Raschke;w. E. van Nostrand