Item#1052: Recombinant tat HIV-1 Bal (E.coli)
Volume/vial: See vial
Diluent: 50mM Tris, 0.1M NaCl, 2mM dtt, pH7.8
Physical State: Frozen Liquid
Stability: At least 6 months at -75°C.
Application: ELISA, Western ELISA, Anti-tat Drug Screening, Immunization, Transcriptional Activation.
Description: Full length (101 amino acids) Recombinant HIV-1 Bal tat produced in the E.coli Expression System.
Purification: This protein was purified by solvent extraction, ion affinity and macro-molecular sieving to approx.95% purity, as determined by SDS-PAGE, reduced.
Specificity: This protein binds to murine monoclonal antibodies of defined epitope specificity and rabbit and human serum polyclonal antibodies in ELISA and Western ELISA.
Biological Activity: Not Done
Endotoxin: Assay pending
Application 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-phosphate buffer (150mM NaCl, 50mM sodiumphosphate, 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 1-10µg/ml range. ELISA and Western ELISA require tat in 10-100ng protein range.
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).
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