Help:Page name

From DrugPedia: A Wikipedia for Drug discovery

[edit] Proteome analysis of membrane of M.tuberculosis can provide novel drug targets

Proteome analysis,involving a combination of 2D electrophoresis, mass spectrometry, and bioinformatics, has now emerged as a robust and efficient strategy for rapid identification of proteins (Jungblut et al., 1996).Immunogenicity of membrane proteins, as reported with certain pathogens, has been attributed to their inherent hydrophobicity and lipid modification (Deres et al., 1989; Akins et al., 1993; Frankenburg et al., 1996). Bioinformatic analysis of the M. tuberculosis genome predicts >65 lipoproteins of ‘cell envelope’ origin, some of which were identified previously as ‘secreted’ proteins, or enzymes involved in cell wall biogenesis. Besides these, there are 17 conserved MmpL and MmpS proteins, and >600 other ‘putative’ membrane proteins. The latter differ in the number of transmembrane hydrophobic segments, and include proteins belonging to the majorfacilitator and ATP-binding cassette (ABC) superfamilies (Tekaia et al., 19 unidentified proteins, three of the known ‘immunodominant’ ones: the 19 and 38 kDa lipoproteins and the 33/36 kDa ‘proline-rich’ protein. Sudhir Sinha, Shalini Arora, K. Kosalai, Abdelkader Namane, Alex S. Pym and Stewart T. Cole have published technique of proteome analysis in their research article Proteome analysis of the plasma membrane of Mycobacterium tuberculosis Comp Funct Genom 2002; 3: 470–483. [1].The technique is as follows:

Isolation and fractionation of mycobacterial cell membranes

3–4 week old bacterial cultures(M.tuberculosisH37Rv & BCG) were harvested and probe-sonicated in a buffer containing protease inhibitors (50 mM Tris,10 mM MgCl2, 1 mM EGTA, 1 mM PMSF, 0.02% NaN3, pH 7.4). The sonicates were centrifuged, initially at 23 000 × g; 3: 470–483to remove cell walldebris and later at 150 000 × g to obtain the membrane sediment. The membrane sediment was resuspended in sonication buffer at a concentration of 10 mg protein/ml, to which precondensed Triton X114 (Bordier, 1981) was added, at a final concentration of 2%. The suspension was then stirred (1 h, 4 ◦C) to obtain the extract in a single phase. Residual insoluble matter was removed by centrifugation at 150 000 × g and the phases were allowed to separate at 37 ◦C in a water bath. The upper (aqueous) and lower (detergent) phases were collected by centrifugation (1000 × g) and ‘back-washed’ three times. Protein in the pooled detergent phase was recovered by precipitation with 5 volumes of chilled acetone. Dried sediment was resuspended in distilled water and stored as lyophilized aliquots.

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE)

A solubilization buffer (7 M urea, 2 M thiourea, 4% CHAPS, 0.3% DTT, 2% carrier ampholytes 3–10 and 40 mM Tris, pH 9.6) recommended for isoelectric focusing (IEF) of membrane proteins (Friso and Wikstrom, 1999) was used. 1 mg lyophilized protein was solubilized in 500µl this buffer by vortexing (30 min). This was followed by centrifugation (12 000 rpm × 10 min) to remove the insoluble matter. 150 µl (∼300 µg protein) or 450 µl (∼900 µg protein) clear supernatant was applied, respectively, to immobilized pH gradient (IPG) strips using the method of ‘in-gel rehydration’ (Gorg et al., 2000). IEF was performed at 20 ◦C in an IEF cell using the following four-step program for 7 cm IPG strips: (a) 0–250 V in 1 h; (b) 250 V constant for 1 h; (c) 250–3000 V in 4 h; and (d) 3000 V constant until 15 kVh. The current limit was set at 50 µA/strip. After IEF, IPG strips were equilibrated sequentially (Gorg et al., 2000) in solutions ‘A’ (0.05 M Tris–HCl, pH 8.8, containing 6 M urea, 30% glycerol, 2% SDS, 1% DTT) and ‘B’ (0.05 M Tris–HCl, pH 8.8, containing 6 M urea, 30% glycerol, 2% SDS,4%iodoacetamide, 0.005% bromophenol blue). Later, each strip was loaded on top of a vertical SDS-polyacrylamide gel and sealed in place with 1% low-melting agarose dissolved in electrode buffer. Molecular mass markers were loaded in a separate well by the side of the strip. Electrophoresis was performed using Laemmli’s (1970) buffer system at a constant current of 20 mA. Proteins were stained with Coomassie brilliant blue R250. Images were acquired by an imaging densitometer.

Peptide mass mapping

For in-gel digestion, sample preparation was performed as described by Shevchenko et al. (1996). Briefly, the Coomassie blue stained spot was excised from the gel, washed, in-gel reduced, S-alkylated with iodoacetamide and digested with bovine trypsin (sequencing grade, Roche Molecular Biochemicals) at 37 ◦C overnight. Peptides were extracted, dried in a SpeedVac and resolubilized in 8µl. 0.1% TFA. ZipTips (Millipore)were used to desalt the samples. Peptide mass mapping was performed on 0.5µl Tryptic digest mixture using α-cyano-4-hydroxycinnamic acid (CHCA, Sigma). The samples were analysed by MALDI–MS on a Voyager DE STR (PerSeptive Biosystems, Framingham, MA, USA) equipped with a nitrogen laser (337 nm). The instrument was operated in the delayed extraction mode with a delay time of 150 ns.

MALDI–MS analysis of 61 spots led to the identification of 32 proteins, 17 of which were new to the M. tuberculosis proteome database. The authors classified 19 of the identified proteins as ‘membrane-associated’; 14 of these were further classified as ‘membrane-bound’, three of which were lipoproteins. The remaining proteins included four heat-shock proteins and several enzymes involved in energy or lipid metabolism.The identity of a low molecular mass protein, which was expressed prominently in M. tuberculosis but not at all in BCG, was revealed as ESAT-6 by peptide mass mapping. The genome of M. tuberculosis H37Rv has five copies of a cluster of genes known as the ESAT-6 (early secretory antigenic target) loci. These clusters contain members of the ESAT-6 gene family (encoding secreted T cell antigens that lack detectable secretion signals), as well as genes encoding secreted, cell-wall associated serine proteases, putative ABC transporters, ATP-binding proteins and other membrane associated proteins (Tekaia et al., 1999).The protein in M. leprae appears in the cell wall fraction (Spencer et al., 2002). Thus there is ‘circumstantial’ evidence for the existence of this protein in the cell membrane.ESAT-6 gene family and other membrane-associated uncharacterized proteins found by this study can serve as novel drug targets,candidates for vaccines or diagnostic probes.

--RajniGarg 14:24, 5 September 2008 (IST)