Figure 1.

(A) Organization of PPR motifs in KRIPP11. (B) Scheme of the overexpression construct for the MBP-KRIPP11 fusion protein emphasizing the His₆ tag at the C-terminus of KRIPP11. TEV protease cleavage site, which leaves two additional residues (GS) at the N-terminus of KRIPP11, is indicated. (C) Overexpression construct for free MBP with a His₆ tag at the C-terminus. (D) SDS-PAGE for MBP-KRIPP11-His₆ fractions from the Ni-NTA column. Supt., supernatant; m, PageRuler™ size marker; IB, inclusion bodies; FT, flow through; three lanes of wash and three of eluate. (E) SDS-PAGE of MBP-KRIPP11-His₆ fractions from the amylose column. FT, two lanes flow through; m, marker; four lanes of wash and four of eluate. (F) 6–12% gradient native polyacrylamide gel showing the monomeric state of KRIPP11. TEV cleavage mixture is shown in the absence and presence of DDM detergent (see text). For this and subsequent figures, uncropped originals of all gel images are available as Supplementary Information (Fig. S2).