Synthesis and secretion of biologically active mouse interferon-beta using a Bacillus subtilis alpha-amylase secretion vector.
Mouse interferon-beta (IFN-beta) cDNA, whose signal sequence had been removed by BAL 31 digestion, was introduced into a Bacillus subtilis secretion vector constructed by using the promoter and signal sequence of the B. subtilis alpha-amylase gene. The resultant chimeric plasmids were transferred into B. subtilis 207-25. Four kanamycin-resistant transformants were selected by both colony hybridization and a new immunoblot method for secretory proteins. They secrete the proteins which cross-react with sheep anti-mouse IFN-beta serum into the culture medium. One of them expressed a high IFN-beta activity as assayed by the L cell and vesicular stomatitis virus system, while the other three showed weak or little IFN activities. Based on our previous study [Ohmura et al., Nucl. Acids Res. 12 (1984) 5307-5319], it was suggested that the secreted IFN molecules are hybrid proteins in which the NH2-terminal region consists of part of the alpha-amylase signal peptide. Nucleotide sequence analysis revealed that plasmid pTUB502, which expressed high IFN activity, is joined to the mouse IFN-beta gene from the codon position 6 of its mature protein. The other three plasmids, pTUB506, pTUB509, and pTUB519, contain the mouse IFN-beta gene from the codon positions 3, 1, and -5, respectively. The NH2-terminal region of the mouse IFN-beta seems to be closely related to its biological activity.
D000818:Animals; D001412:Bacillus subtilis / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D005822:Genetic Vectors; D007370:Interferon Type I / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D000516:alpha-Amylases / Q000235:genetics
D001426:Bacterial Proteins; D007370:Interferon Type I; D000516:alpha-Amylases