Preparations of recombinant HIV-1 p66 antigen to improve the specificity of immune complex transfer enzyme immunoassay of antibody IgG to HIV-1 reverse transcriptase.
Recombinant HIV-1 p66 (rp66, a subunit of reverse transcriptase (RT), a heterodimer of p66 and p51) was produced in Escherichia coli in three different ways. First, rp66 was produced as a part of the fusion protein of lacZ protein and HIV-1 pol protein consisting of three components: protease (p10), RT (p51/p66), and integrase (p31), and was released from the fusion protein by the protease (pol-rp66). Second, rp66 with Ser-Ser at the N-terminus was produced as a fusion protein with maltose-binding protein containing a factor Xa site between the two proteins (MBP-Ser-Ser-rp66) and was released from the fusion protein by factor Xa (Ser-Ser-rp66). Third, rp66 with Met-Gly at the N-terminus was produced in transformed cells (Met-Gly-rp66). The recombinant proteins were purified from sonic extracts of transformed cells by ammonium sulfate fractionation and various column chromatographies. MBP-Ser-Ser-rp66 and Met-Gly-rp66 were readily purified in sufficient amounts for labeling with 2, 4-dinitrophenyl groups and beta-D-galactosidase from E. coli, but pol-rp66 and Ser-Ser-rp66 were not for enzyme-labeling. Ser-Ser-rp66 was not only polymerized but also degraded to considerable extents. The purified preparations were labeled with 2,4-dinitrophenyl groups and beta-D-galactosidase and were tested in immune complex transfer enzyme immunoassay of antibody IgG to HIV-1 RT using serum samples from 600 HIV-1 seronegative and 30 HIV-1 seropositive subjects. Among various combined uses of the two labeled preparations, the uses of 2,4-dinitrophenylated MBP-Ser-Ser-rp66 and pol-rp66 with beta-D-galactosidase-labeled Met-Gly-rp66 showed the highest (99.8%) and the second highest (99.5%) specificities, which were higher than that with the labeled preparations used in the previous study (98. 0%).
Hashinaka
K
K
Department of Biochemistry, Miyazaki Medical College, Kiyotake, Japan.
Nishikata
I
I
Hashida
S
S
Adachi
A
A
Oka
S
S
Ishikawa
E
E
D000595:Amino Acid Sequence; D002845:Chromatography; D000939:Epitopes; D004926:Escherichia coli; D015658:HIV Infections / Q000175:diagnosis* / Q000276:immunology; D054303:HIV Reverse Transcriptase / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D015497:HIV-1 / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods* / Q000592:standards; D007074:Immunoglobulin G / Q000032:analysis / Q000097:blood; D008969:Molecular Sequence Data; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics; D014170:Transformation, Genetic; D001616:beta-Galactosidase / Q000235:genetics
D000939:Epitopes; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D054303:HIV Reverse Transcriptase; D001616:beta-Galactosidase