Cloning of the mspA gene encoding a porin from Mycobacterium smegmatis

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 a ctivity 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 a greement with the predicted mass of mature MspA. Dissociation of the porin by boiling in 80% dimethyl sulphoxide yielded the MspA monomer, which did n ot farm channels any more, Escherichia coil 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 solve nts. These proteins were specifically detected by a polyclonal antiserum th at was raised to purified MspA of M. smegmatis, These results demonstrate t hat the mspA gene encodes a protein of M. smegmatis, which assembles to an extremely stable oligomer with high channel-forming activity, Database sear ches did not reveal significant similarities to any other known protein. So uthern blots showed that the chromosomes of fast-growing mycobacterial spec ies contain homologous sequences to mspA, whereas no hybridization could be detected with DNA from slow growing mycobacteria, These results suggest th at MspA is the prototype of a new class of channel-forming proteins.