Fig. 2.

a Elution profile of CmSP proteases on DEAE-Sephadex A-50 anion exchange chromatography. The partially purified 30–70% ammonium sulfate fraction was loaded onto DEAE-Sephadex A-50 column. The column (3 × 15 cm) was pre equilibrated with 20 mM phosphate buffer pH 7.2. The unbound proteins were washed with same buffer. Gradient elution was carried out using sodium chloride in 20 mM phosphate buffer pH 7.2 (0–0.5 M) and 2 mL fraction were collected at a flow rate of 0.5 mL/min. Protein content of each fractions were measured at 280 nm (---•---) and protease activity (‒‒‒) was assessed using casein as substrate. Fractions showing proteolytic activity were pooled. b Elution profile of CmSP proteases on Sephadex G-100 column chromatography. The proteases from the active fractions of a Sephadex G-100 chromatography was pooled and loaded onto the Sephadex G-100 gel filtration column (1.5 × 120 cm) equilibrated and eluted with 20 mM phosphate buffer pH 7.2. The flow rate was maintained at 15 mL/hr and fractions of volume 1.25 mL were collected every 5 min. The protein content was monitored continuously for each fraction at 280 nm (-----). The proteolytically active fractions (‒‒‒) eluted from Sephadex G-100 chromatography were pooled and stored for further analysis. Inset: Molecular weight marker and protein from Sephadex G-100 c Protein profile of different fractions on SDS-PAGE (15%). CmSP (15 μg in each well) was loaded on SDS-PAGE under denaturing, reducing conditions and the protein bands were visualized by silver staining. Lane MWM-Molecular weight marker (High range), Lane 1—Crude extract, Lane 2–70% ammonium sulphate dialysed fraction, Lane 3—DEAE Sephadex A-50 anion exchange sample, Lane 4—Sephadex G-100 fraction. d Casein zymogram for protease activity (10%, Native PAGE). Lane 1-crude extract, lane 2–70% ammonium sulphate dialysed fraction, lane 3-DEAE A-50 fraction, lane 4-Sephade G-100 fraction, The extracted and purified CmSP were run in 10% native PAGE containing copolymerised casein. The proteins were stained with CBB R-250 and destained with glacial acetic acid/methanol/distilled water (1:3:6). The activity band is observed as a clear colorless area depleted of casein in the gel against the blue back-ground