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Dobos Lab: Native Proteins

Antigen 85

Reagent: Antigen 85 Complex, Ag85, Mixture of Rv3804c (Ag85A, FbpA), Rv1886c (Ag85B, FbpB), and Rv0129c (Ag85C, FbpC)
Default Quantity: 500 µg
Production System: CFPs are precipitated with a 40% saturated solution of ammonium sulfate. The precipitate is suspended and applied to phenyl sepharose HPLC. Antigen 85 is obtained by increasing the pH and eluting with a high concentration of ethylene glycol. If necessary, additional purification is performed using size exclusion chromatography. The purified product is dialyzed against 0.01 M ammonium bicarbonate, lyophilized, and stored at -80°C.
Notes: The Ag85 complex contains three gene products of 30-31 kDa. The individual components of the Ag85 complex (Ag85A, Ag85B and Ag85C) are available for specific research needs, and the default quantity for these is 250 µg.
References:
Wiker H.G. and Harboe M., Microbiol. Rev. 56:648, 1992.


45kDa Glycoprotein

Reagent: Rv1860, 45 kDa glycoprotein, MPT32, ModD, Apa
Default Quantity: 500 µg
Production System: CFPs are precipitated with a 40% saturated solution of ammonium sulfate. The precipitate is suspended and incubated with Conconavalin-A resin, and the 45 kDa is eluted with alpha-methyl mannoside. This fraction is concentrated and dialyzed. A final purification is performed using hydrophobic interaction (phenyl sepharose) HPLC. The purified 45 kDa glycoprotein is lyophilized and stored at -80°C.
Notes: The 45 kDa glycoprotein appears as a doublet by SDS-PAGE. The major component is 45 kDa and the minor component is a 42 kDa degradation product. The mature MPT 32 glycoprotein has a calculated molecular weight of 30.2 kDa, however the abundance of proline residues along with glycosylation causes this molecule to migrate at 45 kDa by SDS-PAGE.
References:
Dobos K.M. et al., Infect. Immun. 63:2846, 1995.
Dobos K.M. et al., J. Bact. 178:2498, 1996.


GroES

Reagent: Rv3418c, GroES, 14 kDa heat shock protein
Default Quantity: 500 µg
Production System: A supernatant from a 40% saturated ammonium sulfate precipitation of CFPs is precipitated with 70% saturated ammonium sulfate. The pellet is suspended, and run over a conA column. The material that does not bind to the column is then resolved by reversed phase HPLC column. Fractions containing the GroES homologue are pooled. Aliquoted samples are stored at -80°C.


Alpha-crystallin

Reagent: Rv2031c, alpha-crystallin, 16 kDa antigen, HspX
Default Quantity: 100 µg
Production System: Whole cell lysate is extracted with 0.1% n-octylthioglucoside in PBS and centrifuged at 27,000 xg for one hour. The supernatant is fractionated by isoelectric focusing (BioRad Rotofor system). Fractions containing 16kDa are pooled and cleaned up using size exclusion chromatography. Pure fractions of 16 kDa are pooled, dialyzed against 0.01 M ammonium bicarbonate, quantified using the BCA protein assay, lyophilized, and stored at -80°C.
References:
Lee B., et al. Infect. Immun. 60:2066, 1992.


PhoS1

Reagent: Rv0934, PhoS1, 38 kDa antigen, Anitgen 5, PstS1
Default Quantity: 250 µg
Production System: The proteins soluble in 40% ammonium sulfate are precipitated with a 70% saturated ammonium sulfate solution. These proteins are suspended in ConA binding buffer and applied to a ConA column. ConA elution buffer, containing alpha-D-mannopyranoside, is used to elute the bound proteins. The bound material is applied to a phenyl sepharose column and eluted using a gradient of decreasing ammonium sulfate. Fractions containing pure 38kDa are pooled and dialyzed. Aliquots are stored at -80°C.
Notes: It should be noted that the cell wall associated and culture filtrate forms of PhoS1 seem to differ in their hydrophobic characteristics. However, we have not yet determined if there is a chemical difference between the cell wall and culture filtrate forms of the PhoS1 homologue. The PhoS1 provided by this contract is purified from culture filtrate.

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