title: Large-scale production and characterization of recombinant human immunodeficiency virus type 1 Nef.
abstract: Sequences encoding the 27K and 25K nef gene products (Nef 27 and Nef 25) were amplified by PCR from a human immunodeficiency virus type 1 infectious clone and subcloned directly into Escherichia coli, yeast and baculovirus expression vectors. The yeast- and baculovirus-derived Nef had native N termini but the expression levels were low. The expression levels of the E. coli-derived glutathione S-transferase-Nef fusion proteins were very high and a major portion was soluble. Large-scale production of E. coli-derived Nef 27 and Nef 25 was carried out by growing recombinant cells in a fermenter under fed-batch conditions followed by affinity purification on glutathione-Sepharose before and after thrombin cleavage. Large quantities of highly purified recombinant Nef proteins have been produced for functional and structural studies. Under non-reducing conditions both Nef 27 and Nef 25 existed as a mixture of monomers, dimers and small amounts of higher oligomers, but when reduced were monomeric. The highly purified Nef proteins had no G protein activities, however Nef 27 was biologically active. When electroporated into uninfected CD4+ T lymphocytes both E. coli-derived Nef 27 and yeast-derived myristylated Nef 27 down-regulated the surface expression of CD4, demonstrating that this method can be used to assess the biological activity of purified recombinant Nef.
authors.affiliation: Biomolecular Research Institute, Parkville, Victoria, Australia.
authors.forename: A A
authors.initials: AA
authors.lastname: Azad
authors.forename: P
authors.initials: P
authors.lastname: Failla
authors.forename: A
authors.initials: A
authors.lastname: Lucantoni
authors.forename: J
authors.initials: J
authors.lastname: Bentley
authors.forename: C
authors.initials: C
authors.lastname: Mardon
authors.forename: A
authors.initials: A
authors.lastname: Wolfe
authors.forename: K
authors.initials: K
authors.lastname: Fuller
authors.forename: D
authors.initials: D
authors.lastname: Hewish
authors.forename: S
authors.initials: S
authors.lastname: Sengupta
authors.forename: S
authors.initials: S
authors.lastname: Sankovich
mesh_terms: D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D015693:Gene Products, nef / Q000096:biosynthesis*; D015754:Genes, nef / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis*; D054311:nef Gene Products, Human Immunodeficiency Virus
chemical_list: D015693:Gene Products, nef; D011994:Recombinant Proteins; D054311:nef Gene Products, Human Immunodeficiency Virus