Figure 2
From: Preparation of cell-permeable Cre recombinase by expressed protein ligation
Semi-synthesis of Cre-R9 by EPL. (A) Expression of Cre-intein-His was induced by the addition of IPTG (lane 3). Cre-intein-His was purified by Ni-NTA chromatography (lane 4). After dialysis, Cre-intein-His was cleaved by MESNA and ligated with CGSGG-R9 peptide at 4°C for 24 h (lane 5). Cre-R9 was isolated from intein-His and CGSGG-R9 peptide by Ni-NTA chromatography (lane 6) and gel filtration (lane 7). Lane 1, size marker. The 12% SDS-PAGE gel was stained with Coomassie Brilliant Blue R-250. (B) The 12.5% SDS-PAGE gel was run for a longer running time, and it separated Cre-R9 from Cre-thioester as the distinct bands (lane 3). Cre was prepared by the DTT-mediated cleavage of Cre-intein-His (lane 2). Lane 1, size marker. The gel was stained with Coomassie Brilliant Blue R–250. The ratio of Cre-R9:Cre-thioester, which was estimated from the integrated peak areas of densitometry analysis of lane 3, was 84:16.