Secretory expression in Escherichia coli and Bacillus subtilis of human interferon alpha genes directed by staphylokinase signals

Molecular & General Genetics : MGG
R BreitlingD Behnke

Abstract

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 alpha 1 (hIFN alpha 1) and hybrid hIFN alpha 1/2 genes to this sak ESU resulted in secretory expression of the two gene products in both Escherichia coli and Bacillus subtilis. While most of the IFN alpha was exported to the periplasmic space of E. coli, about 99% was secreted to the culture medium by recombinant B. subtilis strains. The total yield in E. coli was 1.2 x 10(5) IU/ml. This level of expression and export led to instability of the recombinant strains that was spontaneously relieved in vivo by inactivation of the sak ESU through insertion of an IS1 element. No such instability was observed with B. subtilis although expression and secretion levels reached even 3 x 10(6) IU/ml. Proteolytic degradation of IFN alpha 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 IFN alpha 1 protein purified from B. subtilis culture superna...Continue Reading

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Citations

Oct 1, 1990·Current Opinion in Biotechnology·G Rapoport, A Klier
Jul 15, 2015·Cytokine & Growth Factor Reviews·Nawal Abd El-BakyElrashdy M Redwan
May 3, 2014·Preparative Biochemistry & Biotechnology·Nawal Abd El-Baky, Elrashdy M Redwan
Jun 12, 2016·Microbial Cell Factories·Christopher P LandowskiMarkku Saloheimo
May 17, 2008·Protein Expression and Purification·Anand GhosalkarAradhana Srivastava
Jan 25, 2017·Microbial Cell Factories·Antonia LakowitzRebekka Biedendieck
Mar 1, 1993·Microbiological Reviews·M Simonen, I Palva

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