Figure 4

Purities 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).