LARGE-SCALE PRODUCTION AND CHARACTERIZATION OF RECOMBINANT HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 NEF

Sequences encoding the 27K and 25K nef gene products (Nef 27 and Nef 2 5) were amplified by PCR from a human immunodeficiency virus type 1 in fectious clone and subcloned directly into Escherichia coli, yeast and baculovirus expression vectors. The yeast- and baculovirus-derived Ne f had native N termini but the expression levels were low. The express ion 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 gro wing recombinant cells in a fermenter under fed-batch conditions follo wed by affinity purification on glutathione-Sepharose before and after thrombin cleavage. Large quantities of highly purified recombinant Ne f proteins have been produced for functional and structural studies. U nder non-reducing conditions both Nef 27 and Nef 25 existed as a mixtu re of monomers, dimers and small amounts of higher oligomers, but when reduced were monomeric. The highly purified Nef proteins had no G pro tein activities, however Nef 27 was biologically active. When electrop orated into uninfected CD4(+) T lymphocytes both E. coli-derived Nef 2 7 and yeast-derived myristylated Nef 27 down-regulated the surface exp ression of CD4, demonstrating that this method can be used to assess t he biological activity of purified recombinant Nef.