High yield secretion of human erythropoietin from tobacco cells for ex vivo differentiation of hematopoietic stem cells towards red blood cells

Human erythropoietin (EPO) is a key cytokine in erythropoiesis by regulating differentiation of erythroid progenitor cells into red blood cells (RBCs). Plant cell cultures are considered as promising alternative bioproduction platforms for EPO. To overcome the bottlenecks of low protein productivity and secretion, EPO was expressed in tobacco BY-2 cells with a designer peptide tag, termed (SP)(20) that consists of 20 tandem repeats of a "Ser-Pro " motif. This de novo designed tag directed extensive O-glycosylation on each Pro residue in plant cells and acted as a molecular carrier to promote the extracellular secretion of EPO. To facilitate the establishment of stable and high-expression BY-2 cell lines, EPO molecules were co-expressed with a reporter protein GFP, which could be used as a visual marker to monitor the protein expression during the subculture. The engineered (SP)(20) glycomodule substantially increased the secreted yields of EPO up to 4.31 mu g/mL. The (SP)(20)-tagged EPOs exhibited the expected activity in promoting the proliferation of TF-1 cells, though their EC50 was 12-fold higher than that of EPO standard. The (SP)(20)-tagged EPOs could also stimulate the ex vivo expansion and differentiation of hematopoietic stem cell (CD34(+) cells) towards RBCs.