Enhanced production of recombinant human gastric lipase in turnip hairy roots
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Treatment with 2,4-D induced the development of callus-like structures on Brassica rapa hairy roots grown in liquid medium. The structures probably originated from pericycle cell divisions and developed in a non-concentric manner to give conical organs, which did not separate from the roots, whereas the root cortical and epidermal cell layers degenerated. Various cell types were found within the structures, indicating that these differ from simple calli. They are likely to represent secondary root primordia that fail to elongate as a result of hormonal pressure. Hairy roots transformed with a human gene encoding gastric lipase (hGL) were used to produce the recombinant protein. Lipase activity was measured in the culture medium of these roots. The analysis of N-glycans on hGL produced by hairy roots revealed the presence of paucimannosidic N-glycans. Moreover, when the root cultures were treated with 2,4-D, approximately 2.7 times more lipase activity was measured in the culture medium, compared to untreated root cultures, revealing the higher efficiency of this system for the production of a heterologous protein. In addition, some His-tag epitope was found on EGFP in the medium of 2,4-D-treated roots expressing a his-egfp gene, whereas the same tag was totally degraded in the medium of untreated roots, suggesting a reducing effect of 2,4-D on the release of proteolytic activity in the culture medium. We propose to name this new hairy root-derived protein production process “rhizocalli”.
KeywordsAuxin Brassica Gastric lipase N-Glycosylation Hairy roots
We thank Jean-William Dupuy (Plateforme Protéome de Bordeaux) for the nano-LC-MS/MS analysis, Solène Bassard, Gaëtan Vanier and Philippe Chan (PISSARO proteomic platform, Université de Rouen Normandie) for technical assistance, Carol Robins for corrections to the manuscript, and the European Regional Development Fund (FEDER), the “Région Picardie”, and the Root Lines Technology company for funding part of this project.
All authors contributed to the experiment design. JPEE did the transformation experiment, the lipase PAGE and Western analyses, the lipase assays, analysed and formatted the data and reviewed and edited the manuscript. MB performed the N-glycan analysis, wrote the relevant part of the M&M section, and reviewed and edited the manuscript. MBC initiated the hairy root work and reviewed and edited the manuscript. PL reviewed and edited the manuscript. FG initiated the rhizocalli work, did the cloning and the microscopy experiments, the GFP assay and Western analysis, formatted the data and wrote the paper.
Compliance with ethical standards
Conflict of interest
JPEE, MBC and FG are inventors on patents EP2385130. Method for producing recombinant proteins from plant hairy roots and WO 2016/185122 A1. Method for producing proteins from a plant structure owned by the University of Picardie Jules Verne. MBC is a founder, shareholder and scientific adviser of the company Root Lines Technology which holds the patent licences.
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