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 into 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-terminal 13 amino acids of the cII gene, 8 amino acids resulting from the ligation procedure, and the 180 amino acids that comprise the HIV-2 Nef sequence from the NIH-Z strain. The bacterially expressed Nef protein has been purified to apparent homogeneity on analytical scale (10-20 micrograms) by a combination of sequential detergent extraction, gel filtration, and reversed-phase high-performance chromatography. The expressed Nef protein is highly susceptible to proteolysis (chymotryptic-like activity) and this property accounts for the low yield obtained by gel filtration and RP-HPLC. Larger amounts (> 100 micrograms) of the purified Nef 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 production of polyclonal and monoclonal antibodies. The milder method of purification should facilitate structure-function studies of the Nef protein and its role in the life cycle of HIV.
Du Bois
G C
GC
Nucleic Acid and Protein Synthesis Laboratory, Program Resources, Inc., National Cancer Institute, Frederick Cancer Research and Development Center, Maryland 21701.
Hodge
D R
DR
Hanson
C A
CA
Samuel
K P
KP
Zweig
M
M
Showalter
S D
SD
Papas
T S
TS
D000595:Amino Acid Sequence; D010582:Bacteriophage lambda / Q000235:genetics; D002845:Chromatography; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D019204:GTP-Binding Proteins / Q000235:genetics; D015870:Gene Expression; D015693:Gene Products, nef / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*; D005814:Genes, Viral; D005822:Genetic Vectors; D015483:HIV Antibodies / Q000097:blood; D015658:HIV Infections / Q000276:immunology / Q000382:microbiology; D015498:HIV-2 / Q000235:genetics* / Q000276:immunology; D006801:Humans; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D054311:nef Gene Products, Human Immunodeficiency Virus
D015693:Gene Products, nef; D015483:HIV Antibodies; D011993:Recombinant Fusion Proteins; D054311:nef Gene Products, Human Immunodeficiency Virus; D019204:GTP-Binding Proteins