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

R. G. Hewinson*, D. P. Harris, A. Whelan, W. P. Russell

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

7 Citations (SciVal)

Abstract

We have reported previously (R. G. Hewinson and W. P. Russell, J. Gen. Microbiol. 139:1253-1259, 1993) 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 B- and 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 B- and 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.

Original languageEnglish
Pages (from-to)23-29
Number of pages7
JournalClinical and Diagnostic Laboratory Immunology
Volume3
Issue number1
DOIs
Publication statusPublished - 01 Jan 1996

Keywords

  • Amino Acid Sequence
  • Animals
  • Antigens, Bacterial/biosynthesis
  • Bacterial Proteins/biosynthesis
  • Base Sequence
  • DNA Primers/genetics
  • Escherichia coli/genetics
  • In Vitro Techniques
  • Mice
  • Mice, Inbred C57BL
  • Molecular Sequence Data
  • Molecular Weight
  • Mycobacterium bovis/genetics
  • Protein Sorting Signals/genetics
  • Recombinant Proteins/biosynthesis
  • T-Lymphocytes/immunology

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