Characterization of the gene encoding the immunodominant 35 kDa protein of Mycobacterium leprae
Analysis of the interaction between the host immune system and the intracellular parasite Mycobacterium leprae has identified a 35 kDa protein as a dominant antigen. The native 35 kDa protein was purified from the membrane fraction of M. leprae and termed MMPI (major membrane protein I). As the puri...
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Published in | Molecular microbiology Vol. 16; no. 5; p. 865 |
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Main Authors | , , , , , , , , |
Format | Journal Article |
Language | English |
Published |
England
01.06.1995
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Abstract | Analysis of the interaction between the host immune system and the intracellular parasite Mycobacterium leprae has identified a 35 kDa protein as a dominant antigen. The native 35 kDa protein was purified from the membrane fraction of M. leprae and termed MMPI (major membrane protein I). As the purified protein was not amenable to N-terminal sequencing, partial proteolysis was used to establish the sequences of 21 peptides. A fragment of the 35 kDa protein-encoding gene was amplified by the polymerase chain reaction from M. leprae chromosomal DNA with oligonucleotide primers derived from internal peptide sequences and the whole gene was subsequently isolated from a M. leprae cosmid library. The nucleotide sequence of the gene revealed an open reading frame of 307 amino acids containing most of the peptide sequences derived from the native 35 kDa protein. The calculated subunit mass was 33.7 kDa, but the native protein exists as a multimer of 950 kDa. Database searches revealed no identity between the 35 kDa antigen and known protein sequences. The gene was expressed in Mycobacterium smegmatis under the control of its own promoter or at a higher level using an 'up-regulated' promoter derived from Mycobacterium fortuitum. The gene product reacted with monoclonal antibodies raised to the native protein. Using the bacterial alkaline phosphatase reporter system, we observed that the 35 kDa protein was unable to be exported across the membrane of recombinant M. smegmatis. The 35 kDa protein-encoding gene is absent from members of the Mycobacterium tuberculosis complex, but homologous sequences were detected in Mycobacterium avium, Mycobacterium haemophilum and M. smegmatis. The availability of the recombinant 35 kDa protein will permit dissection of both antibody- and T-cell-mediated immune responses in leprosy patients. |
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AbstractList | Analysis of the interaction between the host immune system and the intracellular parasite Mycobacterium leprae has identified a 35 kDa protein as a dominant antigen. The native 35 kDa protein was purified from the membrane fraction of M. leprae and termed MMPI (major membrane protein I). As the purified protein was not amenable to N-terminal sequencing, partial proteolysis was used to establish the sequences of 21 peptides. A fragment of the 35 kDa protein-encoding gene was amplified by the polymerase chain reaction from M. leprae chromosomal DNA with oligonucleotide primers derived from internal peptide sequences and the whole gene was subsequently isolated from a M. leprae cosmid library. The nucleotide sequence of the gene revealed an open reading frame of 307 amino acids containing most of the peptide sequences derived from the native 35 kDa protein. The calculated subunit mass was 33.7 kDa, but the native protein exists as a multimer of 950 kDa. Database searches revealed no identity between the 35 kDa antigen and known protein sequences. The gene was expressed in Mycobacterium smegmatis under the control of its own promoter or at a higher level using an 'up-regulated' promoter derived from Mycobacterium fortuitum. The gene product reacted with monoclonal antibodies raised to the native protein. Using the bacterial alkaline phosphatase reporter system, we observed that the 35 kDa protein was unable to be exported across the membrane of recombinant M. smegmatis. The 35 kDa protein-encoding gene is absent from members of the Mycobacterium tuberculosis complex, but homologous sequences were detected in Mycobacterium avium, Mycobacterium haemophilum and M. smegmatis. The availability of the recombinant 35 kDa protein will permit dissection of both antibody- and T-cell-mediated immune responses in leprosy patients. |
Author | Brennan, P J Eiglmeier, K Winter, N Britton, W J Rivoire, B Hunter, S W Triccas, J A Pessolani, M C Lim, E M |
Author_xml | – sequence: 1 givenname: N surname: Winter fullname: Winter, N organization: Centenary Institute of Cancer Medicine and Cell Biology, Newtown, New South Wales, Australia – sequence: 2 givenname: J A surname: Triccas fullname: Triccas, J A – sequence: 3 givenname: B surname: Rivoire fullname: Rivoire, B – sequence: 4 givenname: M C surname: Pessolani fullname: Pessolani, M C – sequence: 5 givenname: K surname: Eiglmeier fullname: Eiglmeier, K – sequence: 6 givenname: E M surname: Lim fullname: Lim, E M – sequence: 7 givenname: S W surname: Hunter fullname: Hunter, S W – sequence: 8 givenname: P J surname: Brennan fullname: Brennan, P J – sequence: 9 givenname: W J surname: Britton fullname: Britton, W J |
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Snippet | Analysis of the interaction between the host immune system and the intracellular parasite Mycobacterium leprae has identified a 35 kDa protein as a dominant... |
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SubjectTerms | Amino Acid Sequence Antibodies, Monoclonal Antigens, Bacterial - biosynthesis Antigens, Bacterial - genetics Antigens, Bacterial - isolation & purification Bacterial Proteins - biosynthesis Bacterial Proteins - genetics Bacterial Proteins - isolation & purification Base Sequence Cosmids DNA Primers Gene Expression Regulation, Bacterial Gene Library Genes, Bacterial Genes, Dominant Humans Leprosy - immunology Molecular Sequence Data Molecular Weight Mycobacterium - genetics Mycobacterium - metabolism Mycobacterium leprae - genetics Polymerase Chain Reaction Promoter Regions, Genetic Recombinant Proteins - biosynthesis Restriction Mapping Sequence Homology, Amino Acid T-Lymphocytes - immunology |
Title | Characterization of the gene encoding the immunodominant 35 kDa protein of Mycobacterium leprae |
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