title: Hetero-oligomeric MspA pores in Mycobacterium smegmatis.
abstract: The porin MspA of Mycobacterium smegmatis is a biological nanopore used for DNA sequencing. The octameric MspA pore can be isolated from M. smegmatis in milligram quantities, is extremely stable against denaturation and rapidly inserts into lipid membranes. Here, we show that MspA pores composed of different Msp subunits are formed in M. smegmatis and that hetero-oligomers of different Msp monomers increase the heterogeneity of MspA pores designed for DNA sequencing. To improve the quality of preparations of mutant MspA proteins, all four msp genes were deleted from the M. smegmatis genome after insertion of an inducible porin gene from M. tuberculosis. In the msp quadruple mutant M. smegmatis ML712 no Msp porins were detected and mutant MspA proteins were produced at wild-type levels. Lipid bilayer experiments demonstrated that MspA pores isolated from ML712 formed functional channels and had a narrower conductance distribution than pores purified from M. smegmatis with background msp expression. Thus, the M. smegmatis msp quadruple mutant improves the homogeneity of MspA pores designed for DNA sequencing and might also facilitate the identification and functional characterization of other mycobacterial pore proteins.
authors.affiliation: Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
authors.forename: Mikhail
authors.initials: M
authors.lastname: Pavlenok
authors.affiliation: Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA mnieder@uab.edu.
authors.forename: Michael
authors.initials: M
authors.lastname: Niederweis
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002463:Cell Membrane Permeability; D017353:Gene Deletion; D016680:Genome, Bacterial; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics; D009154:Mutation; D020102:Mycobacterium smegmatis / Q000235:genetics* / Q000254:growth & development; D009169:Mycobacterium tuberculosis / Q000235:genetics; D058608:Nanopores; D018272:Porins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000502:physiology; D055503:Protein Multimerization; D017422:Sequence Analysis, DNA
chemical_list: D008051:Lipid Bilayers; D050505:Mutant Proteins; D018272:Porins; C121838:mspA protein, Mycobacterium smegmatis
keywords: DNA sequencing; assembly; nanopore; outer membrane; porin; protein purification