EXPRESSION OF THE LYSOSTAPHIN GENE OF STAPHYLOCOCCUS SIMULANS IN A EUKARYOTIC SYSTEM

The lysostaphin gene of Staphylococcus simulans was cloned into Escher ichia coli. The 5' end of the gene was modified to include a eukaryoti c start codon, the Kozak expression start site consensus sequence, and an enzyme site to facilitate manipulation of the gene. Transcription of the modified gene in vitro yielded an RNA transcript which, when ad ded to a rabbit reticulocyte cell-free translation system, directed th e synthesis of several products. The largest product, migrating at app roximately 93 kDa, as determined by sodium dodecyl sulfate-polyacrylam ide gel electrophoresis, was probably preprolysostaphin, since it was cleaved in the presence of an S. simulans culture supernatant to yield a polypeptide of a size similar to that of mature lysostaphin. When c anine pancreatic microsomal vesicles were added to the translation sys tem, translocation of the newly synthesized polypeptides occurred, as judged by protection from proteolysis. The gene was also expressed tra nsiently from the human cytomegalovirus promoter in COS-7 cells. Activ e enzyme could be detected in the cell lysate, and the prokaryotic sig nal appeared to target secretion of active enzyme to the culture mediu m. The successful expression of the lysostaphin gene and processing of the precursor to produce active secreted enzyme open up the possibili ty of controlling staphylococcal mastitis by targetting expression of this gene to the mammary glands of transgenic animals.