Molecular and General Genetics MGG

, Volume 217, Issue 2–3, pp 384–391 | Cite as

Secretory expression inEscherichia coli andBacillus subtilis of human interferon α genes directed by staphylokinase signals

  • Reinhard Breitling
  • Dieter Gerlach
  • Manfred Hartmann
  • Detlev Behnke
Article

Summary

A DNA segment covering the signal sequence coding region, the ribosome binding site, and the promoter of the staphylokinase (sak) 42D gene (Behnke and Gerlach 1987) was cloned into pUC19 to form a portable expression-secretion unit (ESU). Fusion of human interferon α1 (hIFNα1) and hybrid hIFNα1/2 genes to thissak ESU resulted in secretory expression of the two gene products in bothEscherichia coli andBacillus subtilis. While most of the IFNα was exported to the periplasmic space ofE. coli, about 99% was secreted to the culture medium by recombinantB. subtilis strains. The total yield inE. coli was 1.2×105 IU/ml. This level of expression and export led to instability of the recombinant strains that was spontaneously relieved in vivo by inactivation of thesak ESU through insertion of an IS1 element. No such instability was observed withB. subtilis although expression and secretion levels reached even 3×106 IU/ml. Proteolytic degradation of IFNα by extracellular proteases was avoided by a combination of constitutive expression and secretion during the logarithmic growth phase and the use of exoprotease-reduced host strains. The IFNα1 protein purified fromB. subtilis culture supernatant was correctly processed, carried the expected 11 amino acid N-terminal elongation that resulted from DNA manipulations and proved to be homogenous in Western blotting experiments. The same recombinant plasmid that directed efficient secretion of hIFNα1 inB. subtilis gave poor yields when introduced intoStreptococcus sanguis.

Key words

Cloning Heterologous gene expression Protein secretion Streptococcus sanguis 

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Copyright information

© Springer-Verlag 1989

Authors and Affiliations

  • Reinhard Breitling
    • 1
  • Dieter Gerlach
    • 1
  • Manfred Hartmann
    • 1
  • Detlev Behnke
    • 1
  1. 1.Zentralinstitut für Mikrobiologie und Experimentelle TherapieAkademie der Wissenschaften der DDRJena

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