Expression and biochemical characterization of human immunodeficiency virus type 1 nef gene product.
nef genes from human immunodeficiency virus type 1 isolates BH10 and LAV1 (lymphadenopathy-associated virus type 1) were expressed in Escherichia coli under the deo operon promoter. The two proteins found in the soluble compartment of the bacterial lysate were purified by ion-exchange column chromatography to apparent homogeneity. Determination of the amino-terminal sequence revealed glycine as the first amino acid in the Nef protein, indicating removal of the initiator methionine during expression in E. coli. Under native conditions, the recombinant Nef protein is a monomer of 23 kilodaltons. In denaturing polyacrylamide gels, however, BH10 and LAV1 Nef proteins migrate as 28 and 26 kilodaltons, respectively. GTP binding and GTPase activity were monitored during Nef protein purification. These activities did not copurify with the recombinant Nef protein from either the BH10 or the LAV1 isolate. Purified recombinant BH10 Nef protein was used as an immunogen to elicit mouse monoclonal antibodies. A series of monoclonal antibodies were obtained which reacted with sequences at either the amino or carboxy terminus of Nef. In addition, a conformational epitope reacting with native BH10, but not LAV1, Nef was isolated.
Kaminchik
J
J
Biotechnology General, Israel Ltd., Kiryat Weizmann, Rehovot.
Bashan
N
N
Pinchasi
D
D
Amit
B
B
Sarver
N
N
Johnston
M I
MI
Fischer
M
M
Yavin
Z
Z
Gorecki
M
M
Panet
A
A
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D003488:Cyanogen Bromide; D004797:Enzyme-Linked Immunosorbent Assay; D020558:GTP Phosphohydrolases / Q000378:metabolism; D015693:Gene Products, nef / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D015497:HIV-1 / Q000201:enzymology / Q000235:genetics* / Q000276:immunology; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D012191:Retroviridae Proteins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D054334:Viral Regulatory and Accessory Proteins / Q000235:genetics*; D054311:nef Gene Products, Human Immunodeficiency Virus
D000911:Antibodies, Monoclonal; D015693:Gene Products, nef; D012191:Retroviridae Proteins; D054334:Viral Regulatory and Accessory Proteins; D054311:nef Gene Products, Human Immunodeficiency Virus; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D003488:Cyanogen Bromide