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.