Expression, purification and biochemical characterisation of the human immunodeficiency virus 1 nef gene product.
The human immunodeficiency virus 1 (HIV-1) nef gene encoded by the HIV-1 isolate lymphadenopathy-associated virus type 1 was expressed in Escherichia coli under the control of the tac promoter. The protein is found mainly in the soluble part of the bacterial lysate; a simple two-column purification scheme has been developed allowing isolation of the recombinant protein without using denaturing agents. Analysis of the circular dichroism spectra reveals that the purified protein is folded and has a helix content of 16% and a beta-pleated sheet content of 31%. GTPase activity and binding of guanine nucleotides were measured for Nef and compared with the results obtained under identical experimental conditions for p21rasC, which represents a typical, well-characterized guanine-nucleotide-binding (GNB) protein. Within the limits of error, native Nef does not show GTPase activity and does not bind guanine nucleotides strongly (association constant, Kass less than 5 x 10(3) M-1). An upper limit for the association constant of Nef for ATP was determined by equilibrium dialysis as 5 x 10(3) M-1. Nef can be autophosphorylated by ATP; under the experimental conditions used, 1-2% of the protein become phosphorylated. Correspondingly, our Nef preparation shows a low, but significant, ATPase activity. In conclusion, Nef is not a member of the GNB protein family, but a possible role as a protein kinase cannot be excluded.
Wolber
V
V
Max-Planck-Institute for Medical Research, Heidelberg, Federal Republic of Germany.
Rensland
H
H
Brandmeier
B
B
Sagemann
M
M
Hoffmann
R
R
Kalbitzer
H R
HR
Wittinghofer
A
A
D000227:Adenine Nucleotides / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D015870:Gene Expression; D015693:Gene Products, nef / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005798:Genes, Bacterial; D006150:Guanine Nucleotides / Q000378:metabolism; D015497:HIV-1 / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D054311:nef Gene Products, Human Immunodeficiency Virus
D000227:Adenine Nucleotides; D015693:Gene Products, nef; D006150:Guanine Nucleotides; D011994:Recombinant Proteins; D054311:nef Gene Products, Human Immunodeficiency Virus; D000251:Adenosine Triphosphatases; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins