Sephadex g 25 fine resin

The intein-mediated cleavage to generate the Cre-thioester and the ligation of the Cre-thioester with polyarginine polypeptides were performed at the same time. The Cre-intein-His protein in cleavage buffer (50 mM NaH₂PO₄ and 250 mM NaCl, pH 8.0) was incubated with 50 mM 2-mercaptoethanesulfonate (MESNA) and a 10-fold molar excess of CGSGG-R9 or CGSGG-dR9 at 4°C for 24 h. To separate Cre-R9 from the Cre-intein-His or intein-His proteins, the reaction mixture was loaded onto a Ni-NTA column equilibrated with lysis buffer, and the flow-through fraction was collected. Cre-R9 was treated with dithiothreitol (DTT) to a final concentration of 10 mM, incubated on ice for 30 min, and then concentrated using a Pierce concentrator 9K (Thermo) at 4°C. Any leftover MESNA and peptides in Cre-R9 were removed by a spin column packed with Sephadex G25 Fine resin GE Healthcare) equilibrated with storage buffer (50 mM NaH₂PO₄, 300 mM NaCl, and 1 mM DTT, pH 8.0).