Secretion of the mycobacterial 19-kilodalton protein by Escherichia coli, a novel method for the purification of recombinant mycobacterial antigens

Abstract

the secretion by Escherichia coli of a recombinant form of the immunogenic protein MPB70 of Mycobacterium bovis when the protein is translated from its native initiation codon. N-terminal sequence analysis of the purified protein revealed that the signal peptide of MPB70 was cleaved by an endopeptidase of E. coli at the same cleavage site as reported for the protein in M. bovis. Since both the Band T-cell antigenicities of the purified recombinant protein were similar to that of the native protein, the 19-kDa antigen of M. bovis was used as a model to test whether the signal peptide of MPB70 could direct the secretion of heterologous proteins in E. coli and whether antigen produced in this way retained antigenicity superior to that of recombinant protein produced as a fusion to glutathione-S-transferase. A chimeric protein was produced in which the signal peptide of MPB70 was fused to the 19-kDa antigen of M. bovis at amino acid residue 23. This chimeric protein was found to be secreted into the periplasm and culture medium of recombinant E. coli, and the signal peptide was cleaved by an endopeptidase of E. coli during secretion. Secretion of the 19-kDa antigen facilitated purification of the antigen by two-stage preparative electrophoresis which gave yields of 2.5 mg of purified, soluble 19-kDa antigen from 2.5 g (wet weight) of E. coli. Antigen purified in this way retained both Band T-cell antigenicities. Moreover, the nonspecific mitogenic activity of the purified 19-kDa antigen was low, while the magnitude of the T-cell response induced by the purified antigen was considerably higher than that observed with purified antigen produced as a fusion protein with glutathione-S-transferase.

Mycobacterial antigen‐specific antibody responses in bovine tuberculosis: an ELISA with potential to confirm disease status

“…One method is the use of molecular biological techniques in the bulk production of recombinant proteins. Mycobacterial proteins have been expressed in both Escherichia coli (Hewinson and others 1996a) and Mycobacterium smegmatis (Garbe and others 1993) but it has often proved difficult to purify the proteins in sufficient quantities to be of practical value. In the present study each recombinant protein was expressed in E coli by using the expression vector p rset , which incorporates a polyhistidine tag into the expressed fusion protein.…”

Molecular Characterization of MPT83: a Seroreactive Antigen of Mycobacterium tuberculosis with Homology to MPT70

“…An ion exchange chromatography fraction of M. bovis AN5 containing MPB83 and MPB70 was obtained as described elsewhere [23]. The cloning and purification of recombinant MPB70 [31,34] and recombinant 19 kDa antigen have been described previously [35]. The purified 19 kDa antigen preparation was found to contain low concentrations of a 36 kDa and 38 kDa doublet on SDS-PAGE.…”

Identification of Mycobacterium tuberculosis signal sequences that direct the export of a leaderless β-lactamase gene product in Escherichia coli

“…This conclusion is supported by previous data demonstrating that the secreted M . bouis protein MPB70 was efficiently secreted into the periplasm of E. coLi transformed with the entire MPB70 gene, including the mycobacterial ribosome-binding site, start codon and signal peptide (Hewinson & Russell, 1993). The MPB70 signal peptide also directed the export of the 19 kDa M .…”

“…bovis antigen in E. coli (Hewinson et al, 1996a). The nature of the selection system used in our experiments will be biased towards the selection of E. coli-compatible signal sequences and provides no information on the presence of atypical signal sequences and/or export pathways in M .…”

“…tuberculosis infection (Hewinson et al, 1996b), and MPB70 is an immunodominant M . bouis antigen (Hewinson & Russell, 1993). protein containing an extended glycine-and proline-rich apolar domain, Pmtbl, Pmlepl, Xmlepl and Xmtb3 constitute a group of exported proteins that are potentially associated with the cell membrane or other compartments of the lipid-rich mycobacterial cell wall (Fig.…”