Cellular Reprogramming Employing Recombinant Sox2 Protein
Figure 1
Purification of recombinant Sox2-TAT fusion protein and reprogramming setup. (a) The recombinant cell-permeant Sox2 fusion protein [22] consists of the full-length Sox2 protein and a carboxy-terminally fused sequence of tags consisting of a nuclear localization sequence (NLS), cell-penetrating peptide TAT, and a histidine-tag (H6) for single-step purification. (b) Schematic representation of the expression and purification procedure and the reprogramming setup used in this study. After expression in E. coli, Sox2-TAT-containing cell lysates are subjected to affinity column chromatography employing Ni-NTA resin. Purified recombinant Sox2-TAT protein is eluted from the column and its reprogramming competency assessed in combination with retroviruses encoding Oct4, Klf-4, and c-Myc. (c, d) Biochemical analysis of Sox2-TAT purification from E. coli. The following fractions were subjected to SDS-PAGE analysis: crude lysate (CL), marker (M), pellet (P), supernatant (SN), flow-through (FT), washing buffer (W), and elution fraction (E). SDS gels were either stained using Coomassie (c) or used for preparation of an immunoblot using anti-Sox2-specific antibody (d).
