The amplified DNA fragments were ligated to pGEM-T Easy vector DNA, yielding recombinant plasmids pGEM-T/Rv3874, pGEM-T/Rv3875 and pGEM-T/Rv3619c, respectively. The DNA fragments corresponding to rv3874, rv3875 and rv3619c genes from recombinant pGEM-T were subcloned in the expression vector pGES-TH-1,
and their identity was confirmed by DNA sequencing (data not shown). E. coli ABT-888 molecular weight BL-21 cells were transformed with recombinant pGES-TH-1, and SDS–PAGE analysis of cell lysates from transformed E. coli showed the expression of proteins that corresponded to the size of GST/Rv3874 (Fig. 2, panel A, lane 3), GST/Rv3875 (Fig. 2, panel A, lane 4) and GST/Rv3619c (Fig. 2, panel B, lane 3). The E. coli cells carrying the parent plasmid (pGES-TH-I) also expressed free GST that migrated to its expected position (30 kDa) in the gel (Fig. 2, panel A and B, lane 2). The absence of major protein bands at these positions with the parent E. coli cells (Fig. 2, panel A and B, lane 1) implied that the major protein bands in transformed E. coli cells were as a result of the expression of additional proteins from the parent or recombinant
plasmids. The identity of the expressed fusion proteins was established by Western immunoblotting with anti-GST and anti-penta His antibodies. There was no reaction with any cellular protein from the negative control (parent E. coli BL-21 cells) (Fig. 2, panel C, D, E, F; lane 1), while selleck inhibitor the GST protein alone, expressed from the parent plasmid (pGES-TH-l), reacted with the anti-GST antibodies and anti-penta His antibodies, as expected (Fig. 2, panel C, D, E, F; lane 2). A major band of reactivity was obtained with anti-GST antibodies for GST-Rv3874, GST-Rv3875 (Fig. 2C; lane 3, 4, respectively), and GST-Rv3619c (Fig. 2E, lane
3), and with anti-penta His antibodies for GST-Rv3874, GST-Rv3875 (Fig. 2D; lane 3, 4, respectively), and GST-Rv3619c fusion proteins (Fig. 2F, lane 3), which corresponded with the major protein band in Coomassie blue-stained gels and to the expected migration positions of the three fusion proteins. The SDS–PAGE analysis of cell-free extracts and pellets of sonicates Fossariinae of induced E. coli cells containing pGES-TH/Rv3874, pGES-TH/Rv3875 and pGES-TH/Rv3619c showed that GST-Rv3874 and GST-Rv3875 proteins were present in the soluble fraction (Fig. 3A, B, lane 1), whereas GST-Rv3619c was present in the pellet, which solublized best in 4 m urea (Fig. 3C, lane 1). To purify the recombinant mycobacterial proteins, the soluble/solublized fractions were loaded on to glutathione-Sepharose affinity matrix and the GST-free mycobacterial proteins were released from the fusion proteins bound to the column matrix by cleavage with thrombin protease. The analysis of eluted fractions by SDS–PAGE showed that the recombinant Rv3874 and Rv3875 proteins were contaminated with another protein of nearly 70 kDa (Fig.