Figure 6.

Leptospira pure rClpP isoforms and its mixture oligomerization. (A) Size exclusion chromatography of pure rClpP isoforms and its mixture. The oligomeric states of pure rClpP isoforms and its mixture were monitored by size exclusion chromatography using an Enrich 650 High-Resolution column. Elution positions of the standard proteins with its molecular weight are indicated in arrows pointing downward. The molecular weight standard protein markers in kiloDalton (kDa) include β-amylase (200), alcohol dehydrogenase (158), albumin (66), carbonic anhydrase (29), and cytochrome C (12.4). The pure rClpP2 and the rClpP isoform mixture were eluted as a tetradecamer (308 kDa), whereas the pure rClpP1 display a broad elution profile (≥308 kDa). A small shoulder peak corresponding to 200 kDa was also seen for pure rClpP1 and rClpP2. (B) Pure rClpP isoforms or its mixture resolved on the native-polyacrylamide gel. The purified rClpP isoforms or its mixture (2 μg) were resolved on a 4–20% native-PAGE and stained with Coomassie-blue. The standard molecular weight marker was run in lane 1 (M). The purified rClpP1 and rClpP2 resolved at around 480 kDa (23 kDa × 21 = 483 kDa) and 308 kDa (22 kDa × 14 = 308 kDa), respectively, whereas the rClpP isoform mixture was resolved at 480 and 308 kDa. (C) Denaturing polyacrylamide gel electrophoresis of pure rClpP1 after glutaraldehyde (C₅H₈O₂) cross-linking. Chemical cross-linking of pure rClpP1 with glutaraldehyde shows distinct higher molecular weight bands that get intensified with time (0–15 min) (D) Denaturing polyacrylamide gel electrophoresis of pure rClpP2 oligomeric subunits after glutaraldehyde cross-linking. Chemical cross-linking of pure rClpP2 with glutaraldehyde (C₅H₈O₂) show distinct higher molecular weight bands in comparison to control (without C₅H₈O₂). (E) Denaturing gel electrophoresis of the rClpP isoform mixture after glutaraldehyde cross-linking. The cross-linked subunits pattern of rClpP isoform mixture is similar to that of pure rClpP isoforms. The denaturing polyacrylamide gels were stained with Coomassie-blue and for clarity; the image obtained is represented after inversion.