Item# 1002: Recombinant tat HIV-1 IIIB
Concentration: See vial
Volume/vial: see vial
Diluent: 0.2 KCl, 5mM DTT, 50mM Tris, pH 8.0
Physical State: Frozen Liquid
Stability: At least 12 months at -75°C
Applications: ELISA, Western ELISA, Anti-tat Drug Screening, Immunization Transcriptional Activation
Description: Full length (2 Exons, 86 amino acids) Recombinant HIV-1 IIIB tat produced in the E. coli Expression System.
Purification: This protein is purified by ion affinity and reverse phase HPLC to >99% purity, as determined by SDS-PAGE and HPLC.
Molecular weight: 12kD
Specificity: This protein binds to murine monoclonal antibodies of defined epitope specificity and human serum polyclonal antibodies in ELISA and Western ELISA.
Endotoxin: Less than 0.01 EU/mg of protein as determined by BioWhittaker Kinetic QCL Kit.
Biological Activity: The biological specificity of this protein was determined by LTR-CAT activation in scrapie-loaded HeLe Cells, and in U373 MG cells in the presence of 100μM chlorochine diphosphate. Tat concentrations in 1μg/ml range produced at least 25 fold increase in CAT activity. This protein inhibits DP4 activity in vitro and DP4-dependent T-cell activation in vivo.
Applications and Instructions for use
Recommended concentrations for use are approximate values. A dose dependent response assay should be performed to determine the optimal concentration for use in specific applications. Dilute tat stock solution in saline-citrate buffer (150mM NaCl, 50mM sodium citrate, pH 6.5) immediately before use. Tat readily oxidizes in buffer solutions which may change its LTR dependent transcriptional activation activity.
Transcriptional activation assays with tat are performed in 1-5μg/ml range. ELISA and Western ELISA require tat in 10-100ng protein range.
Biological Activity of Purified tat Protein
To examine whether purified tat retained biological activity, a modified scrape loading technique was used to introduce the protein into cell monolayers. In this procedure, either a monolayer culture of HeLa cells transfected 40 hr prior with plasmid pU3R-III or a monolayer culture of HeLa cell line that surface of the dish with a rubber policeman in the presence of the purified tat. Following the scraping procedure, cells were centrifuged, replated in fresh media, and CAT assays were performed 4 hr after addition of protein. As graphically elevated in cells that received that tat.
Gene and Gene Products
Structural Proteins: Structural proteins – the products of gag, pol and env genes, which are essential components of the retroviral particle.
Regulatory Proteins: Regulatory proteins – tat and rev proteins of HIV/SIV and tax and rex proteins of HTLVs; essential for viral expression in infected cells.
Accessory Proteins: Accessory proteins – additional (non-regulatory) virion – and non virion-associated proteins produced by HIV/SIV retroviruses: vif, vpr, vpu, vpx, and nef. Although, the accessory proteins are not necessary for viral propagation in tissue culture, they have been conserved in the different isolates; this conservation and experimental observations suggest that their role in vivo is very important.
gag – group-sepecifc antigens or capsid proteins; the precursor is the p55 myristoylated protein, which is processed to p17 (Matrix) p24 (Capsid) and p7 (NucleoCapsid) proteins by the viral protease. Other small proteins are generated from the gag polyprotein.
pol – (p66) generates the viral enzymes protease (p11), reverse transcriptase (p51), endonuclease and integrase (p32) after the processing of a gag-pol precursor polyprotein by the viral protease; gag-pol precursor is produced by ribosome frameshifting.
env – viral glycoproteins produced as a precursor (gp160) and processed to the external glycoprotein (gp120) and the transmembrane glycoprotein (gp41). The mature proteins are held together by noncovalent interactions; as a result substantial amount of gp120 is released extracellularly. The external glycoprotein (gp120) contains the binding site for the CD4 receptor.
tat – transactivator of HIV gene expression; one of the two necessary viral regulatory factors (tat and rev) for HIV gene expression. Two forms are known, tat-1 exon (minor form) of 72 amino acids, and tat-2 exon (major form) of 86 amino acids. The electrophoretic mobility of these two forms in SDS gels is anomalous; they are approximately 16 kD and 14 kD in weight. Low levels of both proteins are found in persistently infected cells. tat is localized primarily in the nucleolus/nucleus; it acts by binding to the TAR RNA element and activating transcription from the LTR promoter. Post-transcriptional effects of tat have been postulated.
rev – the second necessary regulatory factor for HIV expression. A 19 kD phosphoprotein localized primarily in the nucleolus/nucleus, rev acts by binding to RRE and promoting the nuclear export, stabilization and utilization of the viral mRNAs containing RRE.
vif – viral infectivity factor, typically 23 kD; required for the efficient transmission of cell-free virus in tissue culture. In the absence of vif, the produced viral particles are defective, while the cell-to-cell transmission of virus is not affected significantly. It has been reported that the cellular localization is in the Golgi (vif is not found in the virion).
nef – approximately 27 kD non-virion protein found in the cytoplasm of infected cells. Potentially myristoylated and associated with the inner plasma membrane. One of the first HIV proteins to be produced in the infected cells, it is the most immunogenic of the accessory proteins and may be used in the future for diagnosis and staging of the disease. NEF is dispensable and probably suffers counter-selection during ex vivo viral propagation in vivo. Recent evidence suggests that SIV nef is required for viral propagation in vivo.
vpr – virion-associated protein of unknown function found in HIV-1, HIV-2, SIVmac, and SIVmnd; typically 15 kD. May be homologous to vpx. Also called “rap” for rapid.
vpu – protein that promotes extracellular release of viral particles. Found only in HIV-1. Integral membrane phosphoprotein of 16kd; similar to M2 protein of influenza virus. It may be involved in env maturation. It is not found in the virion.
vpx – virion protein of 12 kD found only in HIV-2 infection. (vpx may have some homology with vpr).
Related research paper:
Stimulation of HIV-1-neutralizing antibodies in simian HIV-IIIB-infected macaques
The humoral immune response to HIV type 1 (HIV-1) infection is inadequate in part because of the narrow range of virus-neutralizing antibodies elicited by the initially infecting virus and the failure to recognize subsequently arising virus variants.
Determination of Neutralizing Activity of Antibodies Against HIV-1 IIIB and MN.
A quantitative syncytium-forming microassay was employed for detection of the virus-neutralizing antibody response (13). Neutralization titers were determined by using two HIV-1 strains, HIV-1 IIIB and HIV-1 MN. A virus-syncytial-sensitive clone of CEM cells (CEM-SS) develops quantifiable, adherent syncytia (syncytium-forming units; SFUs) on a background of confluent, normal CEM monolayer in 4–6 days.
Statistical Analysis of Antibody Responses.
Viral antigen-binding antibodies and HIV-1 IIIB- and MN-neutralizing antibody titers were subjected to statistical analysis to determine the significance of changes observed after vaccination with mAb 1F7 or control mAb TEPC 183.
Analysis of Virus Neutralization Potency.
Blood samples from the three monkeys injected with mAb 1F7 and the control monkey injected with TEPC 183 were analyzed before, during, and after the vaccination regimen. Serial dilution of plasma specimens collected at different time points during the observation period were analyzed for virus-neutralizing antibody activity by using a quantitative syncytia-forming microassay (13) and HIV-1 strains IIIB and MN. The neutralization activity for HIV-1 IIIB and for HIV-1 MN was derived from five plasma dilutions at day 0 (prebleed), and four times after day 0 for each macaque.
Antibody neutralization activity for HIV-1 IIIB increased significantly after four inoculations of 1F7 in monkey 149-93 as determined at day 31