Secretion induction of B. rapa rapa hairy root clone expressing IDUA
To understand whether B. rapa rapa hairy root clone carrying IDUA expression vector would show similar secretion behavior as eGFP clone, the setup shown in Table 1 was applied. The construct configuration of both clones was the same, for a secretory protein, the only difference was the recombinant protein. In this sense, with the same empirical configuration of inducers, we were evaluating the protein-dependency in each system by analyzing how each protein was secreted.
In terms of DW biomass, there was a statistically significant reduction of biomass with the samples ’2,4-D’ and ’PVP+2,4-D’, compared to ’no induction’ (Fig. 4A). However, in general terms, the dry weights were in the same range as those for the eGFP clone. This is in line with the general set-up of the secretion induction experiment, in which the biomass for all treatments was propagated using the same B5mod medium. At that point, all bottles were replicates using the same conditions, and then changing the medium for the different treatments of inducers reflects how these affect the secretion of the respective protein.
The secretion of IDUA protein was analyzed in recovered media from the different treatments after the period of induction. The immunodetection protocol for IDUA detection was performed as described in the 2.5.3 section. The ’PVP+2,4-D+KNO3’ (treatment F) produced the highest IDUA secretion, as reflected in the highest value for relative densitometry (Fig. 4B). Additionally, treatments with’ KNO3’ (treatment C) and with ’PVP+2,4-D’ (treatment E) still induced the secretion of detectable amounts of IDUA protein. In treatments E and F, there are lower bands that depict protein degradation. However, we cannot say they present more protein degradation, possibly it is at the same level but there is just more overall IDUA protein present in treatment F.
The results of IDUA secretion obtained by immunodetection were confirmed using an enzymatic assay to measure the activity of secreted IDUA in the culture media from the different treatments (Fig. 4C) . Treatments C, E and F gave the best results in terms of IDUA activity, especially treatment F with PVP, 2,4D and KNO3 which is higher and statistically different than treatments C and D. Therefore, treatment F allows to obtain the highest amount of active IDUA protein.
The treatments of induction affected the morphology of the hairy roots (Supporting Figure 5) . The treatment with ‘PVP+2,4-D+KNO3´, for instance, eliminated the tendency of the hairy roots to grow upwards in the flask when compared to the roots with the ‘no induction´ treatment. PVP and KNO3, when used independently, did not affect the structure of the tissue when compared to the ‘no induction´ treatment. However, in all treatments in which 2,4-D was used, the root tips got swollen and they developed hump-like structures like the ones previously reported by Ele Ekouna (2017) in B. rapa rapa 2,4-D-treated hairy roots; and by Rage et al. (2020) in Nicotiana benthamiana 2,4-D treated roots. For our experiment, the hump-like structures were spotted after 4-6 days in treatments supplemented with 2,4-D. The morphology changes were the same in the case of the eGFP hairy root clone (data not shown).
2,4-D, affects the morphology of the roots by cell wall remodeling. In presence of a high auxin concentration, the cell wall is loosened, and the turgor pressure against the loosened wall leads to elongation. The general auxin-induced elongation mechanism has been explained before and, briefly, it states that the auxins activate the H+ export, lowering the cell wall pH. This provokes disruption of hydrogen bonding between cellulose microfibrils which in turn loosens the cell wall and consequently elongates cells. After the cell wall elongation, there is a consequent influx of water into the vacuole (Taiz, 1994). For the case of hairy roots, the treatment with 2,4-D only affects the cell wall expansion in cortex and epidermis cells as suggested in previous studies (Ele Ekouna et al., 2017b; Rage et al., 2020). Also, this auxin pressure may have stimulated the formation of lateral roots primordia from pericycle cells. The swelling of roots and of the generated hump-like structures was a consequence of cell wall remodeling and elongation that in turn provoked uptake of water by those specific newly formed structures.
Nitrogen is a crucial macroelement for protein synthesis (Scheible et al., 2004). As already demonstrated by previous studies (Häkkinen et al., 2014; Holland et al., 2010), culture media supplemented with nitrate improves the intra- and extracellular levels of recombinant proteins in tobacco hairy roots, mainly by improving the protein synthesis and stabilizing the secreted proteins.
PVP, a water-soluble polymer, has colloidal and stabilizing properties in different cell cultures while being inert physiologically and metabolically (Magnuson et al., 1996). Some studies have reported that intracellular recombinant proteins were not significantly affected by the addition of PVP, nonetheless, due to the protein stabilizing effect of PVP, the stability of those proteins was significantly improved (Martínez et al., 2005; Pham et al., 2012). On the other hand, in older reports, PVP (up to 3g/l) is reported to have an effect on organ culture and growth of plant cells (LaCount et al., 1997; Magnuson et al., 1996; Sharp and Doran, 2001).
In an experiment with Withania somnifera hairy root culture producing a recombinant globular adiponectin (gAd) as a secretory protein, Dehdashti et al. (2020) showed that their MS media supplemented only with PVP (2g/l) stabilized by 5-fold the extracellular protein being secreted when compared to their control with no PVP added. They also reported that a combination of PVP (1 g/l) and KNO3(2 g/l) resulted in the highest extracellular and intracellular gAd production (1877 μg/l and 21.3 μg/g FW, respectively) significantly higher than their control containing no PVP or KNO3. This illustrates that PVP alone can stabilize the protein when it has been secreted to the apoplast, however, it is only with an extra nitrogen source (i.e. KNO3) that the overall protein production is boosted and in turn stabilized by the PVP already in the media. The analysis of the secreted IDUA using an enzymatic assay to measure the activity of the protein has proved that the produced recombinant protein was indeed active. This protein in such a production system has been already characterized in terms of glycosylation, homogeneity and reproducibility (Cardon et al. 2019).
The improvement of the recombinant protein production by hairy roots ofBrassica rapa rapa by culture medium optimization gave promising perspectives regarding the industrial production of lysosomal enzymes by a hairy root platform. Indeed, it was already proven that the culture of hairy roots in large scale bioreactors for the production of recombinant proteins, such as IDUA, is possible(Cardon et al., 2019; Gutierrez-Valdes et al., 2020). Hairy root culture in large scale bioreactors for the production of therapeutic compounds is suitable for GMP industrial purpose thanks to a controlled and sterile environment. Moreover biomass growth and molecule production can be monitored on-line thanks to identified growth-markers (Samabriva´s internal data) to ensure reproducible batches. The optimized culture medium developed in this study could be applied in large scale cultures in order to improve the production of recombinant protein by hairy roots.