Expression and purification of IGF-E5. A: Structure of IGF-E5. This recombinant protein was made by fusing the complete amino acid sequence of the murine IGF-1 to a His Tag (6H) and to an artificial peptide referred as E5-Coil. The latter possesses a strong affinity for a complementary peptide, the K-Coil . These peptides were designed to promote the interaction of protein containing E5-Coil and K-Coil with each other. The secreted IGF-E5 is 152 amino acids in length. B: The same amount of supernatant of clones secreting IGF-E5 after 7 days of batch culture at 30°C were analyzed by western blot using an antibody against the His Tag. The clones were obtained using the conventional procedure. Arrows: position of IGF-E5. Con: supernatant of non-transfected CHO cells. Arrowhead: position of a minor protein component of unkown nature synthetized by some clones. C: Purification of IGF-E5 produced by clone 4-B4 by affinity chromatography on a nickel column. Unbounded protein fraction (i) and purified IGF-E5 (ii) were analyzed by SDS-PAGE and stained using the protein dye Coomassie Fluor Orange. The same protein fractions were also analyzed by western blot using an anti-His Tag antibody. Arrow: position of IGF-E5. MW: position of molecular weight marker.