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 rev ealed heparin-binding activity. This properly was used to purify the N ef-protein by a one-step procedure, yielding about 90% homogenous Nef- protein as evaluated by silver staining. The Nef-protein was soluble w ithout 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 nucl ear localization in persistently HIV-1 infected glioblastoma cells (Ko hleisen et al., 1992), Nef did not show DNA-binding properties by slot blot/hybridization assay and South/Western blot. In nucleotide-bindin g assays a strong autophosphorylation activity with [gamma-P-32]ATP wa s observed. Nef-protein was not a substrate for ADP-ribosylation by ba cterial toxins arguing against C-protein-like activities of Nef Recomb inant Nef did not interact with membranes as shown by the lack of incr eased fluorescence emission of Nef in the presence of liposomes. The r ecombinant Nef-protein obtained by one-step heparin-based purification shares immunological properties with native Nef and should prove usef ul for further studies of Nef function and immunogenicity.