Abstract

For the rapid selection of higher recombinant hirudin producing strain in a methylotrophic yeast Hansenula polymorpha, a multiple gene integration and dose-dependent selection vector, based on a telomere-associated ARS and a bacterial aminoglycoside 3-phosphotransferase (aph) gene, was adopted. Two hirudin expression cassettes (HV1 and HV2) were constructed using the MOX promoter of H. polymorpha and the mating factor $\alpha$ secretion signal of S. cerevisiae. Multiple integrants of a transforming vector containing hirudin expression cassettes were easily selected by using an antibiotic, G418. Hirudin expression level and integrated plasmid copy number of the tested transformants increased with increasing the concentration of G418 used for selection. The expression level of HV1 was consistently higher than that of HV2 under the similar conditions, suggesting that the gene context might be quite important for the high-level gene expression in H. polymorpha. The highest hirudin producing strain selected in this study produced over 96 mg/L of biologically active hirudin in a 500-mL flask and 165 mg/L in a 5-L fermentor.

주제어

#hirudin   #Hansenula polymorpha   #G418 resistance   #multicopy integration   #MOX promoter  

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참고문헌 (21)

1. High efficiency transformation of Escherichia coli with plasmids , Inoue, H.;H. Nojima;H. Okayama , Gene / v.96,pp.23-28, 1990
   
2. Effects of codon usage and vectohost combination on the expression of spinach plastocyanin in E. coli. , Ejdeback, M.;S. Young;A. Samuelsson;G. Karlsson , Protein Exp. Pur. / v.11,pp.17-25, 1997
   
3. A family of telomere-associate ARSs and their funchings for targeted recombination in Hansenula polymorpha DL-1 , Sohn, J. H.;E. S. Choi;H. A. Kang;J. S. Rhee;S. K. Rhee , J. Bacteriol / v.181,pp.1005-1013, 1999
   
4. Simultaneous expression of the S and L surface antigens of hepatitis B, and formation of mixed particles in the methylotrophic yeast , Janowicz, Z. A.;K. Melber;A. Merckelbach;E. Jacobs;N. Harford;M. Comberbach;C. P. Hollenberg , Hansenula polymorpha. Yeast / v.7,pp.431-443, 1991
   
5. Yeast genetics, molecular aspects. In: I. Campbell and J. H. Duffus (ed.) , Johnston, J. R. , Yeast, a Practical Approach / v.,pp.107-123, 1988
   
6. Sambrook, J.;F. Fritsch;T. Maniatis , Molecular Cloning: A Laboratory Manual, 2nd ed. / v.,pp., 1989
   
7. A dominant selection system designed for the copy-number controlled gene integration in Hansenula polymorpha DL-1 , Sohn, J. H.;E. S. Choi;H. A. Kang;M. O. Agaphonov;M. D. Teavanesyan;J. S. Rhee;S. K. Rhee , Appl. Microbiol. Biotechnol. / v.51,pp.800-807, 1999
   
8. transformation of the methylotrophic yeast Hansenula polymorpha by autonomous replication and integration vectors , Roggenkamp, R. O.;H. Hansen;M. Eckart , Mol. Gen. Genent / v.202,pp.302-308, 1986
   
9. Stable muticopy integration of vector sepuences in Hansenula polymorpha , Gatzka, R. U.;Weydemann;Z. A. Janowicz;C. P. Hollenberg , Appl. Microbiol. Biotechnol / v.43,pp.844-849, 1995
   
10. Production of recombinant proteins by methylotrophic yeasts , Hollenberg, C. P.;G. Gellissen , Curr. Opinion in Biotechnol / v.8,pp.554-560, 1997
    
11. DMSO-enhanced whole cell yeast transformation , Hill, J.;K. A. G. Donald;D. E. Griffiths , Nucleic Acid Res. / v.19,pp.5791, 1991
    
12. Gene expression and secretion of the anticoagulant hirudin in Saccharomyces cerevisiae , Sohn, J. H.;S. K. Lee;E. S. Choi;S. K. Rhee , J. Microbiol. Biotechnol / v.1,pp.266-273, 1991
    원문보기 상세보기
13. Influence of the codon following the initial codon on the expression of the lacZgene in Saccharomyces cerevisiae , Looman, A. C.;M. Laude;U. Stahl , Yeast / v.7,pp.157-165, 1991
    
14. Cloning and expression of a cDNA encoding for the anticoagulant hirudin from the bloodsucking leech , Harvey, R. P.;E. Degryse;L. Stefani;F. Schamber,J. -P. Cazenave;M. Courtney;P. Tolstoshev;J. -P. Lecocq , Hirudo medicinalis. Pros. Natl. Acad. Sci. U.S.A. / v.83,pp.1084-1088, 1986
    
15. Influence on the second and third codon on the expression of recombinant hirudin in E. coli. , Degryse, E. , FEBS Lett. / v.269,pp.244-246, 1990
    
16. Production of recombinant hirudin in Hansenula ploymorpha: Variation of gene expression level depends on methanol oxidase and fermentation strategies , Kim, C. H.;H. W. Seo;H. Y. Kim;J. H. Sohn;E. S. Choi;S. K. Rhee , J. Ind. Microbiol. Biotecnol / v.21,pp.1-5, 1998
    
17. DNA Molecules encoding for FMDH conrol region , Hollenberg, C. P.;Z. A. Janowicz , European Patent Application.EPA No.0299108 / v.,pp., 1987
    
18. Gene expression in recombinant yeast , Gellisen, G;C. P. Hollenberg;Z. A. Janowicz , In: A. Smith (ed.), Gene Expression in Recombinant Microorgamisms / v.,pp.195-239, 1994
    
19. High-level secretion of hirudin by Hansenula polymorpha - Authentioc processing of three different preprohirudins , Weydemann, U.;P. Keup;M. Pionitek;A. W. M. Strasser;J. Schweden;G. Gellissen;Z. A. Janowicz , Appl. Microbiol. Biotechnol / v.44,pp.377-385, 1995
    
20. Vectors for rapid selection of intergrants with different plasmid copy numbers in the yeast Hansenula polymorpha DL1 , Agaphonov M. O.;P. M. Trushdina;J. H. Sohn;E. S. Choi;S. K. Rhee;M. D. Teavanesyan , Yeast / v.15,pp.541-555, 1999
    
21. Codon replacement in the PGK1 gene of Saccharomyces cerevisiae: experimental approach to study the role of biased codon usage in gene expression , Hoekema, A.;R. A. Kastelein;M. Vasser;H. A. DeBoer , Mol. Cell Biol. / v.7,pp.2914-2924, 1987
    

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