Identification of novel transmembrane gene sequence and its use for cell-surface targeting of beta subunit of human chorionic gonadotropin.
We identified a 685-nucleotide gene fragment that codes for the transmembrane and cytoplasmic domains of glycoprotein of the LEP strain rabies virus and carried out experiments designed to express a novel fusion protein on the cell surface. The cDNA encoding the membrane anchor sequence was fused in the correct reading frame to the 3' end of the cDNA encoding the beta subunit of human chorionic gonadotropin (beta(h)CG), a secretory glycoprotein that is used as an antigen for a contraceptive vaccine being developed in our laboratory. The fusion gene cassette was placed under the control of a vaccinia virus early promoter and cloned in a host-restricted fowlpox viral vector. The recombinants, when used to infect mammalian cells that do not allow the replication of fowlpox virus, expressed the N-terminal 135 amino acid residues of beta(h)CG anchored in the cell membrane by the 75-amino acid C-terminal sequence derived from rabies virus glycoprotein. This hybrid protein is correctly processed post-translationally and transported efficiently to the plasma membrane of non-permissive cells such that the anchored beta(h)CG molecule retains the correctly folded native antigenic epitope(s).
Gupta
A
A
National Institute of Immunology, New Delhi, India.
Chandrasekhar
S
S
Pal
R
R
Talwar
G P
GP
Singh
O M
OM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D002642:Chick Embryo; D018997:Chorionic Gonadotropin, beta Subunit, Human / Q000235:genetics* / Q000276:immunology; D003001:Cloning, Molecular; D019084:Fluorescent Antibody Technique, Indirect; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D005814:Genes, Viral / Q000235:genetics*; D020662:Guanine Nucleotide Exchange Factors; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011820:Rabies virus / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015678:Viral Structural Proteins / Q000235:genetics*
D018997:Chorionic Gonadotropin, beta Subunit, Human; D020662:Guanine Nucleotide Exchange Factors; D008565:Membrane Proteins; C490771:RGL1 protein, human; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; D019204:GTP-Binding Proteins