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)‐
The unusual eukaryotic
- NSF-PAR ID:
- 10117538
- Publisher / Repository:
- Wiley-Blackwell
- Date Published:
- Journal Name:
- The Plant Journal
- Volume:
- 88
- Issue:
- 6
- ISSN:
- 0960-7412
- Page Range / eLocation ID:
- p. 1038-1045
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Summary O ‐glycosylated peptides (HypGP s) 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. HypGP s 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 (SP 4)18and 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 ofEGFP , compared with anEGFP 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 (SP 4)18and (SP )32modules underwent Hyp‐O ‐glycosylation with arabino‐oligosaccharides and arabinogalactan polysaccharides, respectively, which were essential in facilitating secretion of the fusedEGFP protein. Distinct non‐Hyp‐O ‐glycosylated (SP 4)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 (SP 4)18module andAGP ‐styled (SP )32module is proposed. -
Summary The ability to edit plant genomes through gene targeting (
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