Applied Microbiology and Biotechnology

, Volume 34, Issue 6, pp 756–764 | Cite as

Expression of the human blood coagulation protein Factor XIIIa in Saccharomyces cerevisiae: dependence of the expression levels from host-vector systems and medium conditions

  • Michael Bröker
  • Oskar Bäuml
  • Anke Göttig
  • Jutta Ochs
  • Michaela Bodenbenner
  • Egon Amann
Applied Genetics and Regulation

Summary

The human blood coagulation protein Factor XIIIa (FXIIIa) was expressed in Saccharomyces cerevisiae employing Escherichia coli-yeast shuttle vectors based on a 2-μ plasmid. Several factors affecting high production yield of recombinant FXIIIa were analysed. The use of the regulatable GAL-CYC1 hybrid promoter resulted in higher FXIIIa expression when compared with the constitutive ADCI promoter. Screening for suitable yeast strains for expression of FXIIIa under the transcriptional control of the GAL-CYC1 hybrid promoter revealed a broad spectrum of productivity. No obvious correlation between the expression rate and the genetic markers of the strains could be identified. The medium composition markedly influenced the FXIIIa expression rates. The expression of FXIIIa was strictly regulated by the carbon source. Glucose as the only sugar and energy source repressed the synthesis of FXIIIa, whereas addition of galactose induced FXIIIa expression. Special feeding schemes resulted in a productivity of up to 100 mg FXIIIa/l in shake flasks.

Keywords

Sugar Carbon Source Galactose Broad Spectrum Saccharomyces Cerevisiae 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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References

  1. Abe A, Wada T, Handa H, Nogi Y, Fukasawa T (1988) Efficient usage of a galactose-inducible expression vector for the production of heterologous protein in yeast. Agric Biol Chem 52:2035–2041.Google Scholar
  2. Amann E, Abel K-J, Grundmann U, Okazaki H, Küpper HA (1988a) Synthesis of human FXIIIa in bacterial cells. Behring Inst Mitt 82:35–42.Google Scholar
  3. Amann E, Ochs B, Abel K-J (1988b) Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli. Gene 69:301–315.Google Scholar
  4. Ammerer G (1983) Expression of genes in yeast using the ADCI promoter. Methods Enzymol 101:192–201.Google Scholar
  5. Bitter GA, Egan KM (1984) Expression of heterologous genes in Saccharomyces cerevisiae from vectors utilizing the glyceraldehyde-3-phosphate dehydrogenase gene promoter. Gene 32:263–274.Google Scholar
  6. Board PG (1979) Genetic polymorphisms of the a subunit of human coagulation factor XIII. Am J Hum Genet 31:116–124.Google Scholar
  7. Bröker M (1987) Transformation of intact Schizosaccharomyces pombe cells with plasmid DNA. BioTechniques 5:516–518.Google Scholar
  8. Bröker M, Bäuml O (1989) New expression vectors for the fission yeast Schizosaccharomyces pombe. FEBS Lett 248:105–110.Google Scholar
  9. Bröker M, Ragg H, Karges HE (1987) Expression of human anti-thrombin III in Saccharomyces cerevisiae and Schizosaccharomyces pombe. Biochim Biophys Acta 980:203–213.Google Scholar
  10. Burnette WN (1981) “Western blotting”: electrophoretic transfer of proteins from sodium dodecylsulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 112:195–203.Google Scholar
  11. Cesareni G, Murray JAH (1987) Plasmid vectors carrying the replication origin of filamentous single-stranded phages. In: Setlow JK (ed) Genetic engineering, vol 9. Plenum Press, New York, pp 135–153.Google Scholar
  12. Chung SI, Lewis MS, Folk JE (1974) Relationships of the catalytic properties of human plasma and platelet transglutaminases (activated blood coagulation factor XIII) to their subunit structures. J Biol Chem 249:940–950.Google Scholar
  13. Das S, Kellermann E, Hollenberg CP (1984) Transformation of Kluyveromyces fragilis. J Bacteriol 158:1165–1167.Google Scholar
  14. Grundmann U, Amann E, Zettlmeissl G, Küpper HA (1986) Characterization of cDNA coding for human factor FXIIIa. Proc Natl Acad Sci USA 83:8024–8028.Google Scholar
  15. Hanahan D (1983) Studies on transformation of Escherichia coli with plasmid DNA. J Mol Biol 166:557–580.PubMedGoogle Scholar
  16. Holmes DS, Quigley M (1981) A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem 114:193–197.Google Scholar
  17. Hopkins DJ, Betenbaugh MJ, Dhurjati P (1987) Effects of dissolved oxygen shock on the plasmid stability of recombinant Escherichia coli containing plasmid pKN401. Biotechnol Bioeng 29:85–91.Google Scholar
  18. Ichinose A, Hendrickson LE, Fujikawa K, Davie EW (1986) Amino acid sequence of the subunit of human factor XIII. Biochemistry 25:6900–6906.Google Scholar
  19. Johnston M (1987) A model fungal gene regulatory mechanism: the Gal genes of Saccharomyces cerevisiae. Microbiol Rev 51:458–476.Google Scholar
  20. Karges HE (1984) Blood coagulation factor XIII: determination by clot stability assays. In: Bergmeyer J, Grass M (eds) Methods of enzymatic analysis. Verlag Chemie, Weinheim, pp 400–405.Google Scholar
  21. Karges HE, Clemens R (1988) Factor XIII: enzymatic and clinical aspects. Behring Inst Mitt 82:43–58.Google Scholar
  22. Kingsman SM, Kingsman AJ, Mellor J (1987) The production of mammalian proteins in Saccharomyces cerevisiae. TibTech 5:54–57.Google Scholar
  23. Kleiman MJ, Gingold EB, Stanbury PF (1986) The stability of yeast plasmid pJDB248 depends on growth rate of the culture. Biotechnol Lett 8:225–230.Google Scholar
  24. Kniskern PJ, Hagopian A, Montgomery DL, Burke P, Dunn NR, Hofman KJ, Miller WJ, Ellis RW (1986) Unusually high-level expression of a foreign gene (hepatitis B virus core antigen) in Saccharomyces cerevisiae. Gene 46:135–141.Google Scholar
  25. Laughton A, Gesteland RF (1984) Primary structure of the Saccharomyces cerevisiae GAL4 gene. Mol Cell Biol 4:260–267.Google Scholar
  26. Lee GM, Song KB, Rhee SK, Han MH (1986) Plasmid stability and growth of recombinant Saccharomyces cerevisiae producing hepatitis B virus surface antigen. Biotechnol Lett 8:385–390.Google Scholar
  27. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y.Google Scholar
  28. Mead DJ, Gardner DCJ, Oliver SG (1986) Enhanced plasmid stability of a 2 μ-based recombinant plasmid in diploid yeast. Biotechnol Lett 8:391–396.Google Scholar
  29. Parker C, DiBiasio (1987) Effect of growth rate and expression level on plasmid stability in Saccharomyces cerevisiae. Biotechnol Bioeng 29:215–221.Google Scholar
  30. Purvis IJ, Andrew AJE, Loughlin L, Brown AJP (1987) The effect of alterations within the 3′-untranslated region of the pyruvate kinase messenger RNA upon its stability and translation in Saccharomyces cerevisiae. Nucleic Acids Res 15:7951–7962.Google Scholar
  31. Rinas U, Risse B, Jaenicke R, Abel K-J, Zettlmeissl G (1990) Denaturation-renaturation of fibrin-stabilizing factor XIII a-chain isolated from human placenta. Properties of the native and reconstituted protein. Biol Chem Hoppe-Seyler 371:49–56.Google Scholar
  32. Rothstein RJ (1983) One-step gene disruption in yeast. Methods Enzymol 101:202–211.Google Scholar
  33. Santiago TC, Purvis IJ, Bettany AJE, Brown AJP (1986) The relationship between mRNA stability and length in Saccharomyces cerevisiae. Nucleic Acids Res 14:8347–8360.Google Scholar
  34. Schultz LD, Tanner J, Hofmann KJ, Emini EA, Condra HJ, Jones RE, Kieff E, Ellis RE (1987) Expression and secretion in yeast of a 400-kDa envelope glycoprotein derived from Epstein-Barr virus. Gene 54:113–123.Google Scholar
  35. Sherman F, Fink GR, Hicks JB (1983) Methods in yeast genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y.Google Scholar
  36. Stepien PP, Brousseau R, Wu R, Narang S, Thomas DY (1983) Synthesis of a human insulin gene. VI. Expression of the synthetic proinsulin in yeast. Gene 24:289–297.Google Scholar
  37. Suzuki K, Matsiu K, Ito S, Fujita K, Matsumoto H (1988) Polymorphism of the a subunit of a coagulation factor XIII: evidence for subtypes of the FXIIIa*I and FXIIIa*2 alleles. Am J Hum Genet 43:170–174.Google Scholar
  38. Takahashi N, Takahashi Y, Putnam FW (1986) Primary structure of the blood coagulation factor XIIIa (fibrinolase, transglutaminase) from human placenta. Proc Natl Acad Sci USA 83:8019–8023.Google Scholar
  39. Wieslander L (1979) A simple method to recover intact high molecular weight RNA and DNA after electrophoretic separation in low gelling temperature agarose gels. Anal Biochem 98:305–309.Google Scholar

Copyright information

© Springer-Verlag 1991

Authors and Affiliations

  • Michael Bröker
    • 1
  • Oskar Bäuml
    • 1
  • Anke Göttig
    • 1
  • Jutta Ochs
    • 1
  • Michaela Bodenbenner
    • 1
  • Egon Amann
    • 1
  1. 1.Research Laboratories of Behringwerke AGMarburgFederal Republic of Germany

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