Figure 4From: Expression and purification of ELP-intein-tagged target proteins in high cell density E. coli fermentationPurities obtained by one or two rounds of inverse transition cycling. Two rounds of inverse transition cycling yielded much higher purities, as confirmed by SDS-PAGE analysis. Lane 1: clarified cell lysate; Lane 2: soluble contaminants removed after ELP precipitation; Lane 3: purified EI-β-gal precursor before intein cleavage with one round of inverse transition cycling; Lane 4: products of the EI-β-gal cleaving reaction with starting material shown in lane 3, showing bands corresponding to cleaved EI tag, released β-gal, and remaining EI-β-gal precursor; Lane 5: purified β-gal after removal of the EI tag and uncleaved precursor from material shown in late 4 via one additional round of inverse transition cycling.; Lane 6: purified EI-β-gal precursor before intein cleavage using two rounds of inverse transition cycling; Lane 7: products of the EI-β-gal cleaving reaction with starting material shown in lane 6, showing bands corresponding to EI-β-gal, EI tag, and released β-gal; Lane 8: purified β-gal after removal of the EI tag and uncleaved precursor from material shown in late 7 via one additional round of inverse transition cycling. In both cases, the recovery of cleaved β-gal is very high after cleavage and removal of the ELP-intein tag (compare β-gal bands in Lanes 4 and 5, and in Lanes 7 and 8).Back to article page