Secretory expression in Escherichia coli and Bacillus subtilis of human interferon alpha genes directed by staphylokinase signals.
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 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 hIFN alpha 1 in B. subtilis gave poor yields when introduced into Streptococcus sanguis.
Breitling
R
R
Zentralinstitut für Mikrobiologie und Experimentelle Therapie, Akademie der Wissenschaften, DDR, Jena.
Gerlach
D
D
Hartmann
M
M
Behnke
D
D
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006801:Humans; D007370:Interferon Type I / Q000235:genetics* / Q000378:metabolism; D008666:Metalloendopeptidases / Q000235:genetics; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals / Q000235:genetics
D007370:Interferon Type I; D021382:Protein Sorting Signals; D004247:DNA; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus