Figure 1
From: Cell-free protein synthesis as a novel tool for directed glycoengineering of active erythropoietin
Glycosylation of cell-free synthesized erythropoietin (EPO) by 1D-SDS-PAGE followed by autoradiography. (A) Monitoring of translocation and glycosylation of cell-free synthesized 14C-labeled EPO. Lane 1: EPO in translational mixture (TL), lane 2: EPO in soluble fraction (SN), lane 3: EPO in vesicular fraction (VF), lane 4: EPO after treatment with EndoH, lane 5: EPO after treatment with PNGaseF. The upper EPO bands correspond to the glycosylated protein, whereas the lower band corresponds to the unglycosylated (lane 1–3) or deglycosylated (lane 4 and 5) protein. Different molecular weight between Endo H and PNGase F digestion results from the different cutting of the enzymes. (B) Time-dependent cell-free synthesis of EPO supplemented with free 14C-mannose (lane 1: 0.5 hour; lane 2: 1 hour; lane 3: 2 hours; lane 4: 4 hours; lane 5: 24 hours). 14C-leucine-supplemented translation reactions of EPO (lane 6: 2 hours) and without template (lane 7: 2 hours) were used as control reactions.
