Improving yield of a recombinant biologic in a Brassica hairy root manufacturing process

Abstract

Hairy root systems have proven to be a viable alternative for recombinant protein production. For recalcitrant proteins, maximizing the productivity of hairy root cultures is essential. The aim of this study was to optimize a Brassica rapa rapa hairy root process for secretion of alpha- l-iduronidase (IDUA), a biologic of medical value. The process was first optimized with hairy roots expressing eGFP. For the biomass optimization, the highest biomass yields were achieved in modified Gamborg B5 culture medium. For the secretion induction, the optimized secretion media was obtained with additives (1.5 g/l PVP + 1 mg/l 2,4- d + 20.5 g/l KNO₃ ) resulting in 3.4 fold eGFP secretion when compared to the non-induced control. These optimized conditions were applied to the IDUA-expressing hairy root clone, confirming that the highest yields of secreted IDUA occurred when using the defined additive combination. The functionality of the IDUA protein, secreted and intracellular, was confirmed with an enzymatic activity assay. A > 150-fold increase of the IDUA activity was observed using an optimized secretion medium, compared with a non-induced medium. We have proven that our B. rapa rapa hairy root system can be harnessed to secrete recalcitrant proteins, illustrating the high potential of hairy roots in plant molecular farming.

Keywords: alpha-l-iduronidase; eGFP; hairy root; plant molecular farming; recombinant protein; secretion.

Figure 1

Biomass accumulation, growth behavior, and eGFP secretion of Brassica rapa rapa hairy roots carrying eGFP encoding gene. (a) Biomass accumulation in fresh weight (FW, g/L) with B5, B5mod, and BGM after growth period of 14 days. (b) Biomass accumulation in dry weight (DW, g/L) with B5, B5mod, and BGM after growth period of 14 days. (c) Growth behavior on B5mod medium (Circles: biomass expressed in FW; squares: biomass in DW). (d) Extracellular eGFP yield (mg/L) during the cultivation (only one replicate assayed). Error bars represent the standard deviation of four (a and b) or three (c) biological replicates. Letters (a, b, c) indicate the statistically significant differences (p < 0.01). BGM, Brassica Growth Medium; eGFP, green fluorescent protein

Figure 2

Brassica rapa rapa hairy root green fluorescent protein (eGFP) secretion induction experiments. (a) A contour‐plot based on the model constructed from the empirical secretion data of the first secretion induction experiment (Tables 1 and 2) showing secreted eGFP related to MeJA and KNO₃. (b) A contour‐plot based on the model constructed from the empirical secretion data of the second secretion induction experiment (Tables 1 and 2) showing secreted eGFP related to MeJA and KNO₃. One outlier (N6) was selected. (c) A contour‐plot based on the model constructed from the empirical secretion data of the third secretion induction experiment (Tables 1 and 2) showing secreted eGFP related to KNO₃.

Figure 3

Biomass accumulation and green fluorescent protein (eGFP) secretion in Brassica rapa rapa hairy root clone in the secretion induction verification (a) Experimental set‐up, (b) Biomass accumulation (dry weight [DW], g/L) (c) Amount of secreted eGFP to culture medium after induction period (mg/L) (one‐way analysis of variance, post hoc Tukey HSD, p < 0.01), (d) Secreted eGFP after secretion induction expressed in mg/g DW hairy root biomass. Error bars represent the standard deviation of five biological replicates. Letters (a, b, c, d) indicate statistically significant differences (p < 0.01). PVP, polyvinyl pyrrolidine

Figure 4

Biomass accumulation and α‐l‐iduronidase (IDUA) secretion in Brassica rapa rapa hairy root clone with optimized parameters. (a) Biomass accumulation (dry weight [DW], g/L). Dry weight biomass after secretion induction (Dunnett T3). Asterisks indicate significant differences in levels p < 0.05 (*). Error bars represent the standard deviation of four biological replicates (b) Immunodetection of IDUA protein in equivalent amounts of recovered media from the different treatments. A positive control of a commercial standard protein (+) (ABIN1464142, Antibodies‐online) and a negative control (−) of clean non‐induction media. The red arrow shows the 70.8 kDa band corresponding to the mature nonglycosylated standard protein. In the contiguous table, the relative densitometries of the different treatments are displayed. (A = PVP; B = 2,4‐D; C = KNO₃; D = PVP + 2,4‐D; E = PVP + KNO₃; F = PVP + 2,4‐D + KNO₃; G = No induction.] (c) IDUA activity assay on the culture medium of the different treatments applied. Error bars indicate standard deviations. *Indicates significant difference based on Student test (p < 0.05). Figure S5 . Visual changes in the IDUA‐expressing Brassica rapa rapa hairy root clone after the induction of secretion. (A) Color and geotrophic changes in representative bottles of each treatment. (B) Close‐up photographs of representative pieces of hairy roots after the different treatments. Yellow arrows point out swollen tips and hump‐like structures in the treatments with 2,4‐D. The photographs are augmented four times the actual size. (C) Bright‐field microscopic photography of the hump‐like structures resulting from any treatment in which 2,4‐D was used. (D) Bright‐field microscopic photographies from “No induction” and “PVP + 2,4‐D + KNO₃.” 1 and 2 show the noninduced tissue (water and toluidine blue (TBO), respectively), there is callus/starch (unstained) in some of the tips which might denote possibility of the tissue to keep growing. Blue in the very tips denotes lignified cell walls and the root apical meristem. 3 and 4 show the tissue induced with “PVP + 2, 4‐D + KNO₃“ (water and TBO, respectively). 2,4‐D induced hump‐like structures can be seen as a proliferation of possibly lateral roots primordia from pericyclic cells. Also, the root‐tip vascular cylinders seem to abruptly deviate due to the formation of the formed humps. Additionally, root tips are swollen and there is absence of root apical meristems (i.e. not blue parts as those in the non‐induced root tips).