The N-terminal 22 amino acids encoded by the gene specifying the major secreted protein of vaccinia virus, strain Lister, can function as a signal sequence to direct the export of a foreign protein.
Cells infected with vaccinia virus strain Lister secrete a polypeptide of approximate molecular weight 35,000 (35K) into the medium. Previous studies identified a cleavable, hydrophobic region of 17 amino acids in the 35K protein which could potentially function as a signal peptide to target the protein to the secretory pathway. Here we report the use of the expression-secretion signals derived from the 35K gene to direct export and secretion of a foreign protein. Vaccinia virus recombinants carrying the bacterial chloramphenicol acetyl transferase gene (cat) immediately downstream from the promoter and the N-terminal coding sequences of the 35K gene were constructed. Our studies show that the N-terminal 22 or 42 amino acids of the 35K protein direct efficient secretion of the CAT protein. However, due to a cryptic glycosylation site within CAT, glycosylated protein was secreted, which reduced enzymatic activity. Activity was restored in the presence of tunicamycin. Removal of the glycosylation site by site-directed mutagenesis abolished glycosylation with no effect on secretion, although CAT activity was again reduced, possibly due to an effect on the active site. The results presented here demonstrate the feasibility of using the promoter and the signal sequence of the 35K gene to generate recombinant viruses for overexpression and secretion of foreign proteins.
Patel
A H
AH
Medical Research Council Virology Unit, University of Glasgow, UK.
Subak-Sharpe
J H
JH
Stow
N D
ND
D000818:Animals; D001483:Base Sequence; D001692:Biological Transport / Q000235:genetics; D015500:Chloramphenicol O-Acetyltransferase / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D021382:Protein Sorting Signals / Q000235:genetics* / Q000378:metabolism; D014616:Vaccinia virus / Q000201:enzymology / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D004274:DNA, Recombinant; D021382:Protein Sorting Signals; D014764:Viral Proteins; D015500:Chloramphenicol O-Acetyltransferase