Transport to the cell surface of a peptide sequence attached to the truncated C terminus of an N-terminally anchored integral membrane protein.
Attempts to construct hybrid proteins that are transported to the plasma membrane are frequently unsuccessful because of perturbations in polypeptide folding. In seeking to minimize this problem, we have used the less common type of integral membrane protein, which has an uncleaved signal-anchor domain and an extracellular carboxyl portion, to transport a peptide sequence of interest to the cell surface. A set of plasmids was constructed that contained the gene encoding respiratory syncytial virus glycoprotein G (RSVG) interrupted immediately after one of several proline codons by a synthetic sequence containing unique restriction endonuclease sites and a stop codon. The shortened RSVG gene was flanked by vaccinia virus DNA to permit cloning and expression in a vaccinia virus vector. An open reading frame encoding four copies of the immunodominant repeating epitope of the circumsporozoite protein of Plasmodium falciparum was inserted into the tails of the truncated RSVG genes. Recombinant vaccinia viruses were isolated and shown to express hybrid proteins that reacted with a monoclonal antibody directed to the repeating circumsporozoite epitope. Moreover, immunofluorescence studies indicated that the peptide was on the external cell surface and available to react with antibodies. Expression of the hybrid protein also occurred in rabbits inoculated with the live recombinant vaccinia virus, as demonstrated by the generation of antibodies that bound to P. falciparum sporozoites in vitro.
Vijaya
S
S
National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892.
Elango
N
N
Zavala
F
F
Moss
B
B
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D005796:Genes; D005814:Genes, Viral; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D012136:Respiratory Syncytial Viruses / Q000235:genetics; D014616:Vaccinia virus / Q000235:genetics; D014759:Viral Envelope Proteins; D014763:Viral Matrix Proteins / Q000235:genetics
C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; D014759:Viral Envelope Proteins; D014763:Viral Matrix Proteins