PURIFICATION OF AN ESCHERICHIA-COLI-EXPRESSED NEF PROTEIN FROM THE HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-2

The entire nef gene sequence of HIV-2, NIH-Z strain, has been cloned i nto the pJL6 expression vector and used for the synthesis of a 23-kDa protein in E. coli. The expressed protein is a fusion between the N-te rminal 13 amino acids of the cII gene, 8 amino acids resulting from th e ligation procedure, and the 180 amino acids that comprise the HIV-2 Nef sequence from the NIH-Z strain. The bacterially expressed Nef prot ein has been purified to apparent homogeneity on analytical scale (10- 20 mug) by a combination of sequential detergent extraction, gel filtr ation, and reversed-phase high-performance chromatography. The express ed Nef protein is highly susceptible to proteolysis (chymotryptic-like activity) and this property accounts for the low yield obtained by ge l filtration and RP-HPLC. Larger amounts (>100 mug) of the purified Ne f protein have been produced by a purification procedure that employs sequential detergent extraction, chromatography on Q-Sepharose in the presence of 7 M urea, and chromatography on hydroxylapatite, also in 7 M urea. The purified HIV-2 Nef protein has been used for the producti on of polyclonal and monoclonal antibodies. The milder method of purif ication should facilitate structure-function studies of the Nef protei n and its role in the life cycle of HIV.