Heparin-binding capacity of the HIV-1 NEF-protein allows one-step purification and biochemical characterization.
Recombinant Nef-protein of HIV-1 Bru derived from Escherichia coli revealed heparin-binding activity. This property was used to purify the Nef-protein by a one-step procedure, yielding about 90% homogenous Nef-protein as evaluated by silver staining. The Nef-protein was soluble without denaturing agents. Native folding of Nef was demonstrated with antibodies against conformational epitopes of Nef by a slot blot assay under native conditions. Despite its affinity to heparin and its nuclear localization in persistently HIV-1 infected glioblastoma cells (Kohleisen et al., 1992), Nef did not show DNA-binding properties by slot blot/hybridization assay and South/Western blot. In nucleotide-binding assays a strong autophosphorylation activity with [gamma-32P]ATP was observed. Nef-protein was not a substrate for ADP-ribosylation by bacterial toxins arguing against G-protein-like activities of Nef. Recombinant Nef did not interact with membranes as shown by the lack of increased fluorescence emission of Nef in the presence of liposomes. The recombinant Nef-protein obtained by one-step heparin-based purification shares immunological properties with native Nef and should prove useful for further studies of Nef function and immunogenicity.
Kohleisen
B
B
GSF-Institut für Molekulare Virologie, Neuherberg, Oberschleissheim, FRG.
Gaedigk-Nitschko
K
K
Ohlmann
M
M
Götz
E
E
Ostolaza
H
H
Goni
F M
FM
Erfle
V
V
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002462:Cell Membrane / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D015693:Gene Products, nef / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015483:HIV Antibodies / Q000276:immunology; D015497:HIV-1 / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D051379:Mice; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014407:Tumor Cells, Cultured; D054311:nef Gene Products, Human Immunodeficiency Virus
D000911:Antibodies, Monoclonal; D015693:Gene Products, nef; D015483:HIV Antibodies; D011993:Recombinant Fusion Proteins; D054311:nef Gene Products, Human Immunodeficiency Virus; D006493:Heparin; D004247:DNA