Figure 1
From: Assembly and proteolytic processing of mycobacterial ClpP1 and ClpP2
ClpP1 and ClpP2 interact with E. coli ClpP and inhibit its peptidase activity. (A) Hydrolysis of the Suc-LY-Amc peptide was carried out as described in the Methods section with 5 μg (black triangles) or 10 μg (all other samples) of the indicated purified proteins produced in BL21(DE3) (closed symbols) or in SG1146a cells (open symbols). (B) Size-exclusion chromatography of recombinant ClpP2 purified from BL21(DE3) when expressed as the clpP1-clpP2(his) 6 operon. 500 μg of purified ClpP2(His)6 was loaded on a Superdex 200 10/30 column as described in the Methods section. Arrowheads indicate the elution of molecular mass standards with their molecular mass in kDa. The horizontal bar indicates the fractions that were collected and pooled for measurement of peptidase activity in Figure 1C. (C) Hydrolysis of the Suc-LY-Amc peptide by 10 μg of the recombinant ClpP2(His)6 purified by SEC as described in (B) after 2 (black circles), 17 (black triangles), and 50 (black squares) days of storage at 4°C. (D) Hydrolysis of the Suc-LY-Amc peptide by 10 μg of total extracts of SG1146a cells overexpressing the pET26b plasmid (open circles) or BL21(DE3) cells overexpressing the pET26b plasmid (black circles), the pET26b plasmid carrying the clpP1(his) 6 (black squares) or the clpP2(his)6 (black triangles) open reading frames, or the clpP1-clpP2(his) 6 operon (black diamonds).