Search

• Plant cell culture is a promising production platform for high-quality growth factors. • Hyp- O -glycosylated (SP) 20 tag increases the secreted yields of SCF up to 2.5 μg/mL. • The orientation of the (SP) 20 tag substantially impacts the potency of SCF. • (SP) 20 -tagged SCF stimulates the differentiation of hematopoietic stem cell CD34+ cells. Ex vivo generation of red blood cells (RBCs) from hematopoietic stem cells (HSCs) used for blood transfusion represents one of the focuses in current regenerative medicine. However, massive production of HSCs-based RBCs requires a significant quantity of erythropoietic growth factors, making manufacturing at large scale cost prohibitive. Plant cell culture is proposed to be a promising bioproduction platform for functional human proteins in a safe and cost-efficient manner. This study exploited a proprietary technology, named HypGP engineering technology, for high-yield production of one of the key erythropoietic growth factors--stem cell factor (SCF)--in plant cell culture. Specifically, a designer hydroxyproline (Hyp)- O -glycosylated peptide (HypGP) comprised of 20 tandem repeats of the "Ser-Pro" motif, or (SP) 20, was engineered at either the N-terminus or C-terminus of SCF in tobacco BY-2 cells. The (SP) 20 tag dramatically increased the secreted yields of SCF up to 2.5 μg/mL. The (SP) 20 -tagged SCF showed bioactivity in promoting the proliferation of the TF-1 cell line, although the SCF-(SP) 20 was 8.4-fold more potent than the (SP) 20 -SCF. Both the (SP) 20 -SCF and SCF-(SP) 20 exhibited desired function in stimulating the expansion and differentiation of human umbilical cord blood CD34+ cells towards RBCs. [ABSTRACT FROM AUTHOR]

Summary: The key technical bottleneck for exploiting plant hairy root cultures as a robust bioproduction platform for therapeutic proteins has been low protein productivity, particularly low secreted protein yields. To address this, we engineered novel hydroxyproline (Hyp)‐O‐glycosylated peptides (HypGPs) into tobacco hairy roots to boost the extracellular secretion of fused proteins and to elucidate Hyp‐O‐glycosylation process of plant cell wall Hyp‐rich glycoproteins. HypGPs representing two major types of cell wall glycoproteins were examined: an extensin module consisting of 18 tandem repeats of 'Ser‐Hyp‐Hyp‐Hyp‐Hyp' motif or (SP4)18 and an arabinogalactan protein module consisting of 32 tandem repeats of 'Ser‐Hyp' motif or (SP)32. Each module was expressed in tobacco hairy roots as a fusion to the enhanced green fluorescence protein (EGFP). Hairy root cultures engineered with a HypGP module secreted up to 56‐fold greater levels of EGFP, compared with an EGFP control lacking any HypGP module, supporting the function of HypGP modules as a molecular carrier in promoting efficient transport of fused proteins into the culture media. The engineered (SP4)18 and (SP)32 modules underwent Hyp‐O‐glycosylation with arabino‐oligosaccharides and arabinogalactan polysaccharides, respectively, which were essential in facilitating secretion of the fused EGFP protein. Distinct non‐Hyp‐O‐glycosylated (SP4)18‐EGFP and (SP)32‐EGFP intermediates were consistently accumulated within the root tissues, indicating a rate‐limiting trafficking and/or glycosylation of the engineered HypGP modules. An updated model depicting the intracellular trafficking, Hyp‐O‐glycosylation and extracellular secretion of extensin‐styled (SP4)18 module and AGP‐styled (SP)32 module is proposed. [ABSTRACT FROM AUTHOR]