Cloning, overexpression and activity of <i>B</i>. <i>cereus</i> ATCC 14579 ColA.

<p>(<b>A</b>) ColA is expressed as a 110.1 kDa protein and consists of a 3.3 kDa signal peptide (aa 1–30), a 7.2 kDa propetide (aa 31–92) and a 99.6 kDa C-terminal part (aa 93–960) of ColA. Expression constructs for N-terminally GST-tagged ColA ΔSP (132.8 kDa) and ColA ΔPP (125.6 kDa) were cloned. Glutamic acid (E) 501 in the active center of ColA was exchanged by an alanine (E501A) to create proteolytically inactive ColA. (<b>B</b>) The expression, enrichment and activity of GST-ColA ΔPP^wt and GST-ColA ΔPP^E501A proteins in IPTG-induced <i>E</i>. <i>coli</i> lysates or purified via GST pull down (PD) experiments were analyzed by SDS-PAGE (left panel) and gelatin zymography (middle panel). To purify ColA ΔPP^wt, transformed <i>E</i>. <i>coli</i> (-) were induced by IPTG to stimulate GST-ColA ΔPP^wt expression. After lysing bacteria, GST-ColA ΔPP^wt was bound to GST sepharose and either eluted by glutathione (PD) as a GST fusion protein (GST-ColA ΔPP^wt) or cleaved and eluted with the PreScission protease (PreSc) to obtain the untagged protease ColA ΔPP^wt.