FIGURE 2.

Expression of MspA deletion mutants in the porin mutant M. smegmatis ML16. Comparison of detergent extracts by SDS-polyacrylamide gel electrophoresis is shown. MspA proteins were selectively extracted from the porin mutant M. smegmatis ML16 (ΔmspA, ΔmspC, and ΔmspD) (16) at 100 °C in a buffer containing 0.5% n-octyl-POE, separated on a 10% polyacrylamide gel, and stained with Coomassie Blue. Lane M contains the protein mass marker (Mark12; Invitrogen). The expression vector pMN016 (wt mspA, wt lane), the empty vector pMS2 (no MspA lane), and the mutated mspA genes (pMN016 derivatives, lanes Δ3--Δ11) were constitutively expressed in ML16. The part of the gel containing the bands of octameric MspA is shown. The intensity of the monomeric band was identical for all MspA proteins. The amount of the loop deletion mutants was quantified by image analysis and normalized to wt MspA. For mutants Δ3, Δ5, Δ7, Δ9, and Δ11, expression was 104, 94, 91, 17, and 40% of wt, respectively. The background signal (no MspA) was 0.2% of that of wt MspA.