Generation of antibodies to the signal peptide of the MPT83 lipoprotein of Mycobacterium tuberculosis.
The mpt83 gene (Rv2873) encodes the exported MPT83 lipoprotein of Mycobacterium tuberculosis. The corresponding identical mpb83 gene of Mycobacterium bovis is expressed to varying extents in different substrains of M. bovis Bacille Calmette Guerin (BCG), BCG Tokyo and BCG Moreau being high producers and BCG Danish 1331, a low producer of the MPB83 protein. Immunization with the 13-mer N-terminal part of the signal peptide of MPT83, MINVQAKPAAAASC, coupled to keyhole limpet haemocyanin (KLH) through the added C-terminal cysteine resulted in rapid antibody formation monitored by enzyme-linked immunosorbent assay (ELISA) with free immunizing peptide on the solid phase. In ELISA, with four 20-mer overlapping peptides covering the N-terminal part of the MPT83 sequence, three polyclonal rabbit antisera reacted only with the N-terminal peptide. Antigenic signal peptide could not be detected in sonicates of BCG Tokyo and BCG Moreau. After SDS-PAGE and blotting, the antibodies reacted with sonicates of recombinant Escherichia coli containing the entire mpt83 gene including the signal sequence, but not with the 22 kDa form of native MPB83 purified from BCG culture filtrate. In partition chromatography the recMPT83 partitioned in the water phase while 26 kDa MPB83 in BCG culture filtrate partitioned in the lipid phase confirming that lipidation at the N-terminal cysteine residue occurs after the splitting of the polypeptide chain by signal peptidase II.
Harboe
M
M
Institute of Immunology, The National Hospital, Oslo, Norway.
Whelan
A O
AO
Ulvund
G
G
McNair
J
J
Pollock
J M
JM
Hewinson
R G
RG
Wiker
H G
HG
D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000096:biosynthesis*; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000235:genetics; D005798:Genes, Bacterial; D008074:Lipoproteins / Q000235:genetics / Q000276:immunology*; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000276:immunology*; D021382:Protein Sorting Signals / Q000235:genetics; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology
D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D000939:Epitopes; D008074:Lipoproteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins