TY - JOUR
T1 - Secretion of the mycobacterial 19-kilodalton protein by Escherichia coli, a novel method for the purification of recombinant mycobacterial antigens
AU - Hewinson, R. G.
AU - Harris, D. P.
AU - Whelan, A.
AU - Russell, W. P.
PY - 1996/1/1
Y1 - 1996/1/1
N2 - 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.
AB - 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.
KW - Amino Acid Sequence
KW - Animals
KW - Antigens, Bacterial/biosynthesis
KW - Bacterial Proteins/biosynthesis
KW - Base Sequence
KW - DNA Primers/genetics
KW - Escherichia coli/genetics
KW - In Vitro Techniques
KW - Mice
KW - Mice, Inbred C57BL
KW - Molecular Sequence Data
KW - Molecular Weight
KW - Mycobacterium bovis/genetics
KW - Protein Sorting Signals/genetics
KW - Recombinant Proteins/biosynthesis
KW - T-Lymphocytes/immunology
UR - http://www.scopus.com/inward/record.url?scp=0030042549&partnerID=8YFLogxK
U2 - 10.1128/cdli.3.1.23-29.1996
DO - 10.1128/cdli.3.1.23-29.1996
M3 - Article
C2 - 8770499
AN - SCOPUS:0030042549
SN - 1071-412X
VL - 3
SP - 23
EP - 29
JO - Clinical and Diagnostic Laboratory Immunology
JF - Clinical and Diagnostic Laboratory Immunology
IS - 1
ER -