Cloning of the mspA gene encoding a porin from Mycobacterium smegmatis.

TitleCloning of the mspA gene encoding a porin from Mycobacterium smegmatis.
Publication TypeJournal Article
Year of Publication1999
AuthorsNiederweis M, Ehrt S, Heinz C, Klöcker U, Karosi S, Swiderek KM, Riley LW, Benz R
JournalMol Microbiol
Date Published1999 Sep
KeywordsAmino Acid Sequence, Cloning, Molecular, Escherichia coli, Molecular Sequence Data, Mycobacterium, Mycobacterium smegmatis, Porins, Protein Denaturation, Recombinant Proteins, Sequence Analysis

Porins form channels in the mycolic acid layer of mycobacteria and thereby control access of hydrophilic molecules to the cell. We purified a 100 kDa protein from Mycobacterium smegmatis and demonstrated its channel-forming activity by reconstitution in planar lipid bilayers. The mspA gene encodes a mature protein of 184 amino acids and an N-terminal signal sequence. MALDI mass spectrometry of the purified porin revealed a mass of 19 406 Da, in agreement with the predicted mass of mature MspA. Dissociation of the porin by boiling in 80% dimethyl sulphoxide yielded the MspA monomer, which did not form channels any more. Escherichia coli cells expressing the mspA gene produced the MspA monomer and a 100 kDa protein, which had the same channel-forming activity as whole-cell extracts of M. smegmatis with organic solvents. These proteins were specifically detected by a polyclonal antiserum that was raised to purified MspA of M. smegmatis. These results demonstrate that the mspA gene encodes a protein of M. smegmatis, which assembles to an extremely stable oligomer with high channel-forming activity. Database searches did not reveal significant similarities to any other known protein. Southern blots showed that the chromosomes of fast-growing mycobacterial species contain homologous sequences to mspA, whereas no hybridization could be detected with DNA from slow growing mycobacteria. These results suggest that MspA is the prototype of a new class of channel-forming proteins.

Alternate JournalMol Microbiol
PubMed ID10476028

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