title: Expression of the lysostaphin gene of Staphylococcus simulans in a eukaryotic system.
abstract: The lysostaphin gene of Staphylococcus simulans was cloned into Escherichia coli. The 5' end of the gene was modified to include a eukaryotic 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 added to a rabbit reticulocyte cell-free translation system, directed the synthesis of several products. The largest product, migrating at approximately 93 kDa, as determined by sodium dodecyl sulfate-polyacrylamide 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 canine pancreatic microsomal vesicles were added to the translation system, translocation of the newly synthesized polypeptides occurred, as judged by protection from proteolysis. The gene was also expressed transiently from the human cytomegalovirus promoter in COS-7 cells. Active enzyme could be detected in the cell lysate, and the prokaryotic signal appeared to target secretion of active enzyme to the culture medium. The successful expression of the lysostaphin gene and processing of the precursor to produce active secreted enzyme open up the possibility of controlling staphylococcal mastitis by targeting expression of this gene to the mammary glands of transgenic animals.
authors.affiliation: AFRC Institute for Animal Health, Newbury, Berkshire, United Kingdom.
authors.forename: C M
authors.initials: CM
authors.lastname: Williamson
authors.forename: A J
authors.initials: AJ
authors.lastname: Bramley
authors.forename: A J
authors.initials: AJ
authors.lastname: Lax
mesh_terms: D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods; D004285:Dogs; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005057:Eukaryotic Cells / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic; D000882:Haplorhini; D008248:Lysostaphin / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D011817:Rabbits; D013210:Staphylococcus / Q000201:enzymology* / Q000235:genetics; D014158:Transcription, Genetic
chemical_list: D008248:Lysostaphin