Expression and secretion of interferon-alpha 1 by Streptomyces lividans: use of staphylokinase signals and amplification of a neo gene.
A gene coding for mature human interferon, IFN-alpha 1, fused to the expression and secretion signals of a staphylokinase gene (sak) derived from Staphylococcus aureus phage 42D, was inserted into the Streptomyces promoter probe vector pIJ487. Streptomyces lividans transformed with the recombinant plasmid (pMG341) secreted biologically active IFN-alpha 1 into the culture medium. Expression of the IFN-alpha 1 gene was at least on the translational level directed by the sak signals since numerous upstream stop codons would have prevented the formation of a fusion protein. Long-term continuous chemostat cultivation under various limitation conditions was used to select clones with an IFN-alpha 1 yield increased about 60-100-fold (1-2 x 10(5) IU/ml). The increase in IFN-alpha 1 formation was accompanied by spontaneous amplification of the adjacent neo gene, but not of the remaining plasmid DNA. Examination of the DNA sequence around the endpoints of the amplified region revealed almost identical stem-loop structures followed by an octanucleotide direct repeat.
Noack
D
D
Central Institute for Microbiology and Experimental Therapy, Academy of Sciences of the G.D.R., Jena.
Geuther
R
R
Tonew
M
M
Breitling
R
R
Behnke
D
D
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004274:DNA, Recombinant / Q000378:metabolism; D005784:Gene Amplification; D005796:Genes; D006801:Humans; D007375:Interleukin-1 / Q000096:biosynthesis / Q000235:genetics*; D008666:Metalloendopeptidases / Q000235:genetics*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000096:biosynthesis*; D013204:Staphylococcus Phages / Q000235:genetics; D013211:Staphylococcus aureus / Q000235:genetics; D013302:Streptomyces / Q000235:genetics*; D014158:Transcription, Genetic
D004274:DNA, Recombinant; D007375:Interleukin-1; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D004262:DNA Restriction Enzymes; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus