Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.6 (thromboplastin)
 13,278 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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%).
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PMID: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. 1090 70

An ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) of antibody immunoglobulin G (IgG) to human immunodeficiency virus type 1 (HIV-1) has been developed using recombinant HIV-1 reverse transcriptase (rRT) as antigen. However, some disadvantages were noted in the use of rRT as antigen: rRT was produced only with low efficiency in widely used strains of Escherichia coli using a rather long DNA fragment (3,012 bp) of the whole HIV-1 pol gene, and it was impossible to produce fusion proteins of RT for simple purification, since rRT is a heterodimer of p66 and p51. In this study, recombinant HIV-1 p51 and p66 with Ser-Ser at the N termini (Ser-Ser-rp51 and Ser-Ser-rp66) were produced in E. coli as fusion proteins with maltose binding protein containing a factor Xa site between the two proteins and were purified after digestion with factor Xa. Ser-Ser-rp51 was produced in larger amounts and purified in higher yields with less polymerization than Ser-Ser-rp66. Polymerized Ser-Ser-rp66 tended to be precipitated on mercaptoacetylation for conjugation to beta-D-galactosidase (used as a label) and showed higher nonspecific and lower specific signals in an immune complex transfer enzyme immunoassay of antibody IgG to HIV-1 than Ser-Ser-rp51. The signals for serum samples of HIV-1-seropositive subjects by immune complex transfer enzyme immunoassay of antibody IgG to HIV-1 using Ser-Ser-rp51 as antigen (Y) were well correlated to those obtained using rRT as antigen (X) (log Y = 0.99 log X + 0.23; r = 0.99). Thus, the use of rp51 as antigen was advantageous over that of rp66 and rRT in an immune complex transfer enzyme immunoassay of antibody IgG to HIV-1.
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PMID:Recombinant p51 as antigen in an immune complex transfer enzyme immunoassay of immunoglobulin G antibody to human immunodeficiency virus type 1. 1106 8