Intracellular expression of hIFN alpha genes in Escherichia coli and Bacillus subtilis directed by staphylokinase signals.
Portable expression units for intracellular formation of heterologous proteins in Escherichia coli and Bacillus subtilis were constructed by inserting the transcription and translation initiation signals of the staphylokinase sak42D gene into the polylinker of plasmid pUC18. Fusions with ATG-gene cassettes coding for mature human interferons (hIFN) alpha 1 and alpha 2 resulted in intracellular expression of both proteins in E. coli. The 20 fold lower yield of hIFN alpha 2 was not due to unfavorable mRNA secondary structure formation as ruled out by constructing a hybrid hIFN alpha 1/alpha 2 gene. Intracellular expression of IFN alpha 1 in B. subtilis reached 6 x 10(4) IU/ml. Nuclease S1 mapping of transcriptional start sites revealed differential promoter usage in E. coli and B. subtilis. In E. coli transcription from the sak42D promoter was drastically reduced by by transcription initiating from upstream lac and tet promoters. In contrast, in B. subtilis transcription proceeded exclusively from the sak42D promoter.
Breitling
R
R
Zentralinstitut für Mikrobiologie, Jena, Germany.
Gase
K
K
Behnke
D
D
D001412:Bacillus subtilis; D001483:Base Sequence; D015153:Blotting, Western; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D015964:Gene Expression Regulation, Bacterial; D007370:Interferon Type I / Q000096:biosynthesis* / Q000235:genetics; D008666:Metalloendopeptidases / Q000502:physiology*; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic
D007370:Interferon Type I; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus