Expression in yeast of a cDNA clone encoding a transmembrane glycoprotein gp41 fragment (a.a. 591-642) bearing the major immunodominant domain of human immunodeficiency virus.
A cDNA clone corresponding to the gp41 gene fragment nucl. 7573-7730 of the human immunodeficiency virus type 1 (HIV-1) was selected from a random HIV-1 genomic library expressed in yeast. This clone encodes a 52-residue long peptide (amino acid (a.a.)) 591-642) bearing the major immunodominant domain (a.a. 598-609) of the HIV-1 transmembrane glycoprotein gp41. Expression of the recombinant peptide pSE-env591-642 was driven by the alpha-mating factor leader sequence contained in a plasmid pSE-x allowing the synthesis and secretion of foreign gene product in Saccharomyces cerevisiae. Time-course analysis of the secretion into culture medium revealed an optimal production of the glycoprotein fragment at 28-30 h with no observable cytotoxicity. The secreted peptide is highly glycosylated with NH2-terminal heterogeneity probably due to different post-translational modifications. The secreted peptide shows an extreme antigenicity since in ELISA assays, as few as 5 microliters/well of crude supernatant are sufficient to obtain a strong detection by monoclonal antibodies or by 100% of sera from HIV-infected individuals. The purified glycopeptide pSE-env591-642 binds to a monoclonal antibody directed against the immunodominant epitope (a.a. 603-609) with an affinity similar to that of the complete glycoprotein gp160 (Kd values within the 10(-10) M range) and with a 100-fold higher affinity than that of a linear peptide fragment SP-env584-609. These results indicate that overexpression in yeast can efficiently provide an abundant source of highly antigenic gp41 protein fragment pSE-env591-642 which retains the antigenic properties of the native gp160 protein. Such a recombinant peptide should therefore be considered as a good candidate for antigen in HIV detection tests.
Gairin
J E
JE
Laboratoire de Physiologie Nerveuse, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France.
Madaule
P
P
Traincard
F
F
Barrès
E
E
Rossier
J
J
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors; D015483:HIV Antibodies / Q000276:immunology; D015488:HIV Antigens / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D015700:HIV Envelope Protein gp41 / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D015497:HIV-1 / Q000235:genetics / Q000276:immunology*; D016056:Immunodominant Epitopes / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics
D004279:DNA, Viral; D015483:HIV Antibodies; D015488:HIV Antigens; D015700:HIV Envelope Protein gp41; D016056:Immunodominant Epitopes; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D004247:DNA