Molecular Cloning, Expression, Purification and Immunological Characterization of Three Low-Molecular Weight Proteins Encoded by Genes in Genomic Regions of Difference ofMycobacterium Tuberculosis

Abstract

The aim of this study was to clone, express and purify three major antigenic proteins, i.e. Rv3874, Rv3875 and Rv3619c, encoded by genes located in regions of difference of Mycobacterium tuberculosis and characterize them for immunogenicity in rabbits. The respective genes were amplified using gene‐specific primers and genomic DNA of M. tuberculosis by polymerase chain reaction. The amplified DNA were cloned into pGEM‐T Easy and subcloned into pGES‐TH‐1 vector for high‐level expression in Escherichia coli and efficient purification. The results showed that the three fusion proteins, i.e. glutathione‐S‐transferase (GST)‐Rv3874, GST‐Rv3875 and GST‐Rv3619c, were expressed at high levels and were purified (free of the GST fusion partner) to homogeneity using glutathione‐Sepharose and Ni‐NTA agarose affinity matrix after cleavage of the column‐bound fusion proteins by thrombin protease. The purified recombinant Rv3874, Rv3875 and Rv3619c proteins were immunogenic and induced antigen‐specific antibodies in rabbits. Testing of the rabbit sera with overlapping synthetic peptides showed that the antibodies were induced to several epitopes that were scattered throughout the sequence of each protein. These results show immunogenicity of all the proteins for inducing antigen‐specific antibodies in rabbits and demonstrate the usefulness of pGES‐TH‐1 vector for obtaining purified recombinant proteins of M. tuberculosis for immunological characterization.

Molecular Cloning, Expression, Purification and Immunological Characterization of Proteins Encoded by Regions of Difference Genes of Mycobacterium tuberculosis

“…testing of sera with individual peptides of each protein demonstrated that rabbit antibodies recognized several linear epitopes that were scattered throughout the sequence of each protein (Hanif et al, 2010a). Interestingly, previous studies using pools of synthetic peptides have shown that all of these proteins (Rv3874, Rv3875 and Rv3619c) are major T cell antigens in humans, and the linear T cell epitopes were scattered throughout the sequence of these proteins Mustafa et al 2000Mustafa et al , 2008.…”

“…The results showed that the recombinant Rv3874 and Rv3875 proteins were contaminated with another protein of nearly 70 kDa, whereas Rv3619c protein was nearly homogeneous (more than 95% pure). The partially purified Rv3874 and Rv3875 proteins were further purified on Ni-NTA agarose affinity matrix, and the analysis of eluted fractions showed the presence of a single sharp band in SDS-PAGE gels, which suggested that Rv3874 and Rv3875 preparations became free of the 70-kDa contaminant and were nearly homogeneous (more than 95% pure) (Hanif et al, 2010a). These results further strengthen the suggestion that pGES-TH-1 is useful for high level expression and efficient purification of recombinant mycobacterial proteins (Ahmed et al, 2003;.…”

“…On the other hand, GST-Rv3874 and GST-Rv3875 proteins were present in the soluble fraction that also contained E. coli protein of 70 kDa, which was capable of binding to glutathioneSepharose column nonspecifically, and was eluted from the column along with Rv3874 and Rv3875. However, the subsequent use of Ni-NTA matrix efficiently removed the contaminating E. coli protein and made the recombinant Rv3874 and Rv3875 proteins homogeneously pure (Hanif et al, 2010a). The immunogenicity of all the three pure recombinant proteins (Rv3874, Rv3875 and Rv3619c) was evaluated in rabbits.…”

“…Rv3874, Rv3875 and Rv3619c, using the pGES-TH-1 expression vector (Hanif et al, 2010a). The complete cloning, expression and purification strategy for these proteins is shown in figure 1, by using Rv3875 as an example ( Fig.…”

“…The immunogenicity of all the three pure recombinant proteins (Rv3874, Rv3875 and Rv3619c) was evaluated in rabbits. The animals were immunized with the purified RD proteins and the sera were tested for antibody reactivity with the full-length recombinant proteins, pools of synthetic peptides covering the sequence of each protein and their individual peptides, as described previously (Hanif et al, 2010a). In brief, polyclonal antibodies were raised in rabbits against the purified and GST-free Rv3874, Rv3875 and Rv3619c recombinant proteins by emulsifying purified proteins (50 μg/ml) with an equal volume of incomplete Freund's adjuvant and injected intramuscularly in the right and left thigh.…”