Abstract：
: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.
journal_name
J Virol Methodsjournal_title
Journal of virological methodsauthors
Kohleisen B,Gaedigk-Nitschko K,Ohlmann M,Götz E,Ostolaza H,Goni FM,Erfle Vdoi
10.1016/0166-0934(96)02049-6subject
Has Abstractpub_date
1996-06-01 00:00:00pages
89-101issue
1eissn
0166-0934issn
1879-0984pii
0166093496020496journal_volume
60pub_type
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