Product Specifications: 

Item# 105-10: Recombinant M Tuberculosis ESAT-6 Antigen (E.coli)

 Concentration: 2mg/vial

 Mass/vial: 1mg

 Volume/vial: 500ul

 Diluent: 50mM NaPO4, pH 7.0, 0.1M NaCl , 0.05% Sod. Sarcosyl >98%

 Purity: >98%

 Stabilizer: None

 Preservative: None

 Storage: -75°C

 Physical State: Liquid, sterile filled

 Stability:  2 year at -75°C

 Applications: ELISA, Western ELISA, TB, Diagnostics.

 Description: M Tuberculosis ESAT-6 Antigen produced in the E. coli expression system.

 Purification: Purified by ion affinity and UF concentration solvent extraction to >98% purity as determined by SDS-PAGE.

 Molecular Weight: 13kD

Specificity: This ESAT-6 protein reacts with human TB serum in ELISA and Western ELISA.

 Biological Activity: Not determined

 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.

ELISA and Western ELISA require 10-100ng protein depending on the nature and affinity of the detection reagent. Human serum polyclonal antibodies yield titers of 1:1000 or greater at 100ng of immobilized protein under standard ELISA conditions.

Region of difference 1 to 3 gene

Glossary

 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

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

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

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

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

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

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

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

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

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

vpx – virion protein of 12 kD found only in HIV-2 infection. (vpx may have some homology with vpr).

Articles related to ESAT-6

The ESAT-6 Protein of Mycobacterium tuberculosis Interacts with Beta-2-Microglobulin (β2M) Affecting Antigen Presentation Function of Macrophage

ESAT-6, an abundantly secreted protein of Mycobacterium tuberculosis (M. tuberculosis) is an important virulence factor, inactivation of which leads to reduced virulence of M. tuberculosis. ESAT-6 alone, or in complex with its chaperone CFP-10 (ESAT-6:CFP-10), is known to modulate host immune responses; however, the detailed mechanisms are not well understood. The structure of ESAT-6 or ESAT-6:CFP-10 complex does not suggest presence of enzymatic or DNA-binding activities. Therefore, we hypothesized that the crucial role played by ESAT-6 in the virulence of mycobacteria could be due to its interaction with some host cellular factors. Using a yeast two-hybrid screening, we identified that ESAT-6 interacts with the host protein beta-2-microglobulin (β2M), which was further confirmed by other assays, like GST pull down, co-immunoprecipitation and surface plasmon resonance. The C-terminal six amino acid residues (90–95) of ESAT-6 were found to be essential for this interaction. ESAT-6, in complex with CFP-10, also interacts with β2M. We found that ESAT-6/ESAT-6:CFP-10 can enter into the endoplasmic reticulum where it sequesters β2M to inhibit cell surface expression of MHC-I-β2M complexes, resulting in downregulation of class I-mediated antigen presentation. Interestingly, the ESAT-6:β2M complex could be detected in pleural biopsies of individuals suffering from pleural tuberculosis. Our data highlight a novel mechanism by which M. tuberculosis may undermine the host adaptive immune responses to establish a successful infection. Identification of such novel interactions may help us in designing small molecule inhibitors as well as effective vaccine design against tuberculosis.

M. tuberculosis is a dangerous and highly successful pathogen that has evolved several mechanisms to manipulate the host immune regulatory network. Proteins secreted by M. tuberculosis play important roles in virulence. One such protein is ESAT-6, which is secreted along with its chaperone CFP-10. Despite a host of studies highlighting modulation of immune responses by ESAT-6, there have not been many that identified host proteins interacting with ESAT-6. We have now found that the host protein β2M interacts very specifically with ESAT-6 at its C-terminal region. The soluble ESAT-6:CFP-10 complex was found to be trafficked into the endoplasmic reticulum, and treatment with recombinant ESAT-6:CFP-10 or the over-expression of ESAT-6 reduced cell surface expression of β2M and molecules which remain associated with it like HLA-I. Recombinant ESAT-6:CFP-10 was also found to reduce classical and cross presentation of peptide antigens by MHC-I molecules. In summary, our data indicate that interaction between ESAT-6 and β2M can reduce the levels of available free β2M that associate with HLA/MHC-I molecules. This could be an interesting mechanism by which M. tuberculosis inhibits classical and cross presentation of peptide antigens in order to prevent or delay the onset of anti-mycobacterial adaptive immune responses.