(228aj) Production Improvement of Butyrylcholinesterase (BChE) in Rice Cell Lines

Toxic nerve agents have been used as chemical weapons since they can disrupt the mechanism by which nerves transfer signals in the nervous system. Toxic nerve agents act by inhibiting the enzyme acetylcholinesterase which is responsible for the breakdown of acetylcholine (ACh) in the synapse. ACh gives the signal for muscles to contract, preventing them from relaxing so the intoxicated person will lose control of body function which can lead to death.

Some antidotes are being produced to combat poisoning by toxic nerve agents. One of them is the human blood protein, butyrylcholinesterase (BChE), which acts as a bioscavenger for organophosphorous nerve agents. BChE belongs to the family of serine hydrolases and can form a tetramer from four identical subunits (each 85 kDa) interacting with each other via a four-helix bundle at the C-termini. Although the tetramer has longer circulatory half-life, each monomer is fully active independent of its oligomerization state. Recombinant BChE has been produced in several expression systems such as mammalian cells (Lockridge et al.1997; Parikh et al. 2011; Xue et al. 2011; Ilyushin et al. 2013), silkworm (Wei et al. 2000), milk of transgenic goats (Huang et al. 2007), insect cells (Brazzolotto et al. 2012), tobacco leaves (Schneider et al. 2014; Geyer et al. 2010) and most recently in rice cell culture (Corbin et al. 2016). However, the expression of active BChE is not easily obtained.

Our approach is the production of recombinant BChE in rice cell culture using the rice alpha amylase 3D (RAmy3D) expression system. Plant cell culture is being used as a platform for the production of biopharmaceuticals because of its safety and cost-effectiveness in the manufacturing and purification of recombinant proteins. BChE has been produced successfully in rice cell culture using the rice RAmy3D (Corbin et al. 2016). The RAmy3D system allows the production of the recombinant protein under the control of an inducible promoter which is activated by sugar starvation and a signal peptide targets the protein for secretion. However, accumulation of the BChE protein into the culture medium has been challenging, perhaps because of an inefficient excretion of the recombinant BChE presumably due to the large size of the protein or perhaps due to BChE degradation by proteases.

In this study, 8 stable transgenic rice cell lines were analyzed for the expression of BChE. The quantity of recombinant BChE produced was measured at the 1, 3, 6, 9, 12 and 15 days post-induction (dpi) in both the culture media (secreted) and within the plant cell aggregate (homogenization). The time course analysis revealed that at 6 dpi the major amount of the protein was found in the callus while for the secreted protein the highest level was obtained between 6 to 9 dpi depending on the cell line.

In order to enhance the accumulation of the secreted protein in the media culture some biopolymers were added. Gelatin, polyethyleneglycol (PEG) and polyvinylpyrrolidone (PVP) were added to the culture medium at two different concentrations 10 g/L and 15 g/L, respectively. The mechanism of action of the biopolymers as stabilizers is not clear yet however a hypothesis is that they are target of proteases rather that the recombinant protein and it helps to block the degradation of the protein of interest (Kwon et al. 2002 ). Our results revealed that the best results were achieved by the 16-10 cell line and the results discussed below are regarding this line. The control used was the cell line induced with the media without sugar and 0 g/L biopolymer. The levels of protein at 6 dpi in the media reached up to 35 µg BChE/ g fresh weight (FW) calli (32 fold compared to control) when 10g/L gelatin was added. At the concentration of 15 g/L gelatin results were similar to 10 g/L gelatin at 6 dpi the levels detected reached 32 µg BChE/ g fresh weight (FW) calli (28 fold compared to control).

Interestingly with the addition of 10 g/L PEG, 9 dpi was the best day of production of BChE in the medium reaching levels up to 45 µg BChE/ g FW calli (21 fold compared to control). In the case of the addition of 15 g/L PEG, it produced 17 µg BChE/ g FW calli at 9 dpi (8 fold compared to control).

On the other hand, PVP did not help to increase the level of BChE found in the culture medium. In fact the levels were similar or lower than the control.

In the calli homogenization it was observed that the addition of either 10 g/L or 15 g/L gelatin or PEG didnâ??t increases the levels of BChE detected, suggesting that the effect of the biopolymer is just in the excreted protein . All results mentioned were statistically significant (p<0.05).

In summary, these data suggest that the best biopolymers used were 10 g/L gelatin and 10 g/L PEG. These results were confirmed by Western blot analysis under denaturing conditions and the monomer of 85kDa was observed in the media culture starting at 6 dpi. Protease activity analysis is being done to establish if the improvement of the BChE accumulation in the media culture is because of the protease enzymes present in the media. Moreover, glycan analysis will identify the glycosylation pattern in either secreted or cell associated BChE.