Large fragments of nef-protein and gp110 envelope glycoprotein from HIV-1. Synthesis, CD analysis and immunoreactivity.
Chemical synthesis of large peptide fragments (from 18 to 66 amino acid residues long) of the gp110 envelope glycoprotein and of nef-protein from HIV-1 was achieved by the solid phase method. Stepwise assembling of the peptide chains was carried out automatically on 4-(oxymethyl)-phenylacetamidomethyl resin using the N-alpha-butyloxycarbonyl amino acids with benzyl-based side chain protecting groups. Two elongation protocols were used depending on the peptide chain length: a standard cycle, mainly characterized by a single coupling step (Boc-amino acid symmetrical anhydride in dimethylformamide), and an optimized one for large peptides, based on a double coupling strategy (Boc-amino acid symmetrical anhydride first in dimethylformamide, then in dichloromethane). Final cleavage of the peptide from the solid support was carried out by anhydrous hydrogen fluoride and crude peptides were purified by C18 reverse phase medium pressure liquid chromatography after molecular filtration. Characterization of the purified peptides was done by analytical HPLC, amino acid content determination, and circular dichroism analysis both in polar (H2O) and in non-polar (TFE) environments. Immunoreactivity of anti-nef positive sera from HIV-1 infected patients by ELISA with the synthetic peptides was investigated. The results showed four major antigenic regions of nef-protein and mainly the immunodominance of the N- and C-termini of the molecule. Several of these peptides should prove to be useful for both diagnosis and vaccination purposes.
Sabatier
J M
JM
UDC, CNRS URA 37-INSERM UR 172, Laboratory of Biochemistry, Faculty of Medicine North, Marseilles, France.
Fontan
G
G
Loret
E
E
Mabrouk
K
K
Rochat
H
H
Gluckman
J C
JC
Montagnier
L
L
Granier
C
C
Bahraoui
E
E
Van Rietschoten
J
J
D000595:Amino Acid Sequence; D002845:Chromatography; D002942:Circular Dichroism; D004797:Enzyme-Linked Immunosorbent Assay; D015693:Gene Products, nef / Q000032:analysis*; D015488:HIV Antigens / Q000032:analysis*; D015497:HIV-1 / Q000032:analysis*; D006801:Humans; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D014759:Viral Envelope Proteins / Q000032:analysis*; D054334:Viral Regulatory and Accessory Proteins / Q000032:analysis*; D054311:nef Gene Products, Human Immunodeficiency Virus
D015693:Gene Products, nef; D015488:HIV Antigens; D014759:Viral Envelope Proteins; D054334:Viral Regulatory and Accessory Proteins; C055004:gp 110 env protein, Human immunodeficiency virus; D054311:nef Gene Products, Human Immunodeficiency Virus