Hairy-root organ cultures for the production of human acetylcholinesterase
© Woods et al; licensee BioMed Central Ltd. 2008
Received: 30 June 2008
Accepted: 23 December 2008
Published: 23 December 2008
Human cholinesterases can be used as a bioscavenger of organophosphate toxins used as pesticides and chemical warfare nerve agents. The practicality of this approach depends on the availability of the human enzymes, but because of inherent supply and regulatory constraints, a suitable production system is yet to be identified.
As a promising alternative, we report the creation of "hairy root" organ cultures derived via Agrobacterium rhizogenes-mediated transformation from human acetylcholinesterase-expressing transgenic Nicotiana benthamiana plants. Acetylcholinesterase-expressing hairy root cultures had a slower growth rate, reached to the stationary phase faster and grew to lower maximal densities as compared to wild type control cultures. Acetylcholinesterase accumulated to levels of up to 3.3% of total soluble protein, ~3 fold higher than the expression level observed in the parental plant. The enzyme was purified to electrophoretic homogeneity. Enzymatic properties were nearly identical to those of the transgenic plant-derived enzyme as well as to those of mammalian cell culture derived enzyme. Pharmacokinetic properties of the hairy-root culture derived enzyme demonstrated a biphasic clearing profile. We demonstrate that master banking of plant material is possible by storage at 4°C for up to 5 months.
Our results support the feasibility of using plant organ cultures as a successful alternative to traditional transgenic plant and mammalian cell culture technologies.
Bioscavenging of organophosphate (OP) by human cholinesterases (ChEs) is emerging as a promising medical intervention for prophylaxis and post-exposure treatment against chemical warfare nerve agents and pesticides, meeting considerable success in pre-clinical studies [1, 2]. ChEs are very efficient in sequestering OPs that become esterified to a serine residue at the active site. This covalent bond is very stable and in the case of certain OPs is further stabilized by subsequent "aging" reactions. With the phosphorylated enzymes having negligible reactivation rates, ChEs are effectively "single-use molecular sponges" requiring the application of stoichiometric rather than catalytic doses for effectiveness. Thus a production system capable of supplying the forecasted demand for large amounts of active ChEs is needed.
Several strategies for production of ChEs were evaluated. Of the two ChEs in humans, only the serum enzyme butyrylcholinesterase (BChE) can be obtained from natural sources, and large-scale purification efforts from outdated blood-banked human plasma were demonstrated [2–4]. Expression in recombinant systems is the only way for producing the physiological target of OPs, acetylcholinesterase (AChE), which is abundant in muscle and nerve tissues but is normally absent from serum. Several mammalian-based recombinant production systems were described including engineered cell cultures [e.g. [5–7]] and the milk of transgenic goats . As an alternative to these systems that are confronted with being supply-restricted, of limited scalability, high cost and risk of human pathogen contamination, we have introduced plants as a production system for human AChE . We carefully optimized the expression constructs , and purification protocols  and demonstrated that plant derived AChE-RER from Nicotiana benthamiana, which retains all of the catalytic properties of a mammalian-derived enzyme and furthermore that plant-derived AChE-RER is capable of completely ameliorating all of the gross clinical symptoms and some of the long-term molecular consequences implicated in OP poisoning .
Despite their promise, there are currently some concerns among regulatory agencies and the public at large, regarding the use of transgenic plants (grown in open fields or in greenhouses) for the production of protein pharmaceuticals . Among the raised issues is that of environmental containment (both in term of transgene escape and inadvertent contamination of non-transgenic plant material). A further perceived difficulty is the regulatory uncertainty whether the lack of tightly controlled growth conditions typical of plant cultivation can satisfy the strict requirements of good manufacturing practice. In this context plant cell or organ cultures grown in bioreactors may prove more adept at clearing the regulatory hurdles associated with plant-based heterologous production systems while maintaining their most important advantages – inexpensive medium consisting of salts and sugar and devoid of mammalian proteins, growth factors and hormones; equivalent purification costs; and unmatched biosafety bearing minimal risk of human pathogens and prions .
While several plant cell lines are available for use, more organized organ culture, such as "hairy root" cultures may present additional benefits e.g. genetic and biochemical stability and faster growth rates resulting in larger mass/medium ratios [14, 15]. Hairy root cultures are obtained by Agrobacterium rhizogenes mediated transformation of plant tissue (explants). A. rhizogenes is a common phytopathogenic and naturally-transforming soil bacterium (for a review see Guillon et al. . It induces neoplastic growth and differentiation of infected plant tissue to form "hairy roots" by activation of genes on a DNA fragment (T-DNA) that is transferred from the bacterial Ri plasmid and integrated into the plant nuclear genome. The transformed plant tissue quickly grows into a highly branched mass in a medium consisting of simple salts and sucrose and useful compounds such as secondary metabolites or recombinant proteins can be recovered from the medium or extracted from the plant tissue .
Here we demonstrate the feasibility of producing human AChE-RER in hairy root cultures derived from transgenic N. benthamiana plants expressing the protein via A. rhizogenes mediated transformation. Hairy root lines were screened for level of expression of AChE-RER and the protein was subsequently purified and its biochemical properties studied and its circulation half-life determined.
Cloning, Tissue Culture and Initial Screening
For initial screening, root samples (100 mg) were lysed in 3 vol of extraction buffer (50 mM Tris, pH 8, 1 M NaCl, 1% Triton X-100) using a FastPrep machine (Qbiogene). Lysates were then clarified by centrifugation at 14,000 × g for 10 min at 4°C. Supernatants were removed and enzymatic activity of AChE-R was measured (see below).
Determination of Growth Kinetics and 4°C storage protocol
Liquid culture growth rate and maximum culture density were determined for hairy roots lines expressing AChE-RER as well as WT roots. MS liquid medium (500 mL) was spiked with 1.5 g hairy root tissue that had been grown on solid media for 2 weeks. Roots were removed from liquid culture and dried briefly on paper towels at the corresponding time points. For AChE purification experiments, hairy root cultures were typically harvested between 4–6 weeks following culture initiation, ensuring maximum culture density. For 4°C storage experiments, roots were grown on 1.5% agar (Sigma) slants containing MS salts and 3% sucrose and allowed to grow at room temperature for 3 weeks in the dark. Slants were then moved to 4°C. At the indicated times, slants were removed and roots were sterilely transferred to 50 mL of liquid MS medium and cultured on a shaker at RT for 3 weeks. Roots were then removed, patted-dry, inspected and weighed.
Purification of Human Acetylcholinesterase
All purification steps were preformed at 4°C. After 4–6 weeks of growth remaining media was removed from the growth flask and roots were washed once with 500 mL extraction buffer (10% sucrose, 5 mM MgCl2, 15 mM Na2S2O5 in PBS, pH 8.0). Root samples were disrupted in a commercial blender with 2 volumes of fresh extraction buffer and subjected to centrifugation at 20,000 × g for 20 minutes to pellet cellular debris. The supernatant was filtered first with Miracloth (Calbiochem) followed with a Grade 50 filter paper (2.7 μm, Whatman). The clarified supernatant was then diluted with PBS to two times initial volume to reduce the metabisulfite concentration and processed by affinity chromatography using 8 mL procainamide-agarose (Sigma) in a 2.5 cm I.D. Econo-column (Bio-Rad). The column was washed with 80 mL PBS and the bound enzyme eluted with 60 mL elution buffer (0.2 M acetylcholine chloride in PBS) into 2 mL fractions. Fractions displaying AChE activity (2–18) were pooled, ammonium sulfate was added to 55%, and the suspension incubated for 1 hr. The protein pellet obtained by centrifugation (20,000 × g, 15 min) was re-suspended in 20 mL buffer 1 (20 mM Na2PO4/NaHPO4, 20 mM NaCl, pH 7.4), dialyzed extensively against buffer 1 for 12 hr in a 50 kDa MWCO cellulose dialysis tubing (Spectrum), and concentrated in a Macrosep 10 kDa MWCO concentrator (Pall) to a final volume of 3 mL. Sodium azide (0.02%) was added and solution was stored at 4°C until further use.
Biochemical characterization of AChE-R
Enzymatic activity of AChE-R was measured using acetylthiocholine iodine (ATChI, Sigma) as the substrate in a SpectraMax 340PC spectrophotometer (Molecular Devices) by the method of Ellman  as previously described . The specific activity of pure preparations of plant-derived AChE (~3000 U/mg protein ) was used to convert activity protein equivalent. Total protein levels were derived using the Bio-Rad Protein Assay Reagent (Bio-Rad) with BSA as the standard. The Michaelis constant (KM) was determined by measuring and plotting AChE activity as a function of ATChI concentration and non-linear regression analysis (Prism software, GraphPad). Inhibition curves were generated by plotting residual AChE activity (measured in the presence of 1 mM ATChI) as a function of inhibitor's concentration using the following acetylcholinesterase inhibitors: neostigmine bromide (Sigma), 1,5-bis(allyldimethylammoniumphenyl)pentan-3-one dibromide (BW, Sigma), diethyl p-nitrophenyl phosphate (Paraoxon, Sigma) and the butyrylcholinesterase specific irreversible inhibitor, tetra-isopropyl pyrophosphoramide (ISO-OMPA, Sigma).
Protein samples were resolved by SDS-PAGE and visualized using a Silver Snap II kit (Pierce) according to manufacturer's instructions.
Groups of five 6–8 week old male FVB/N mice were injected with 30 U of hairy root-derived AChE-RER in PBS (100 μL) or 100 μL PBS as vehicle control. Blood samples (25 μL) were drawn by tail vein knick. Serum was separated from clotted blood by centrifugation (6,000 × g, 30 min, 4°C). Serum samples were assayed for AChE activity in the presence of 50 μM of the butyrylcholinesterase-specific inhibitor Iso-OMPA. Data derived from AChE-injected mice was normalized to data derived from vehicle-treated mice.
Results and Discussion
Creation of hairy root cultures
Previously we described the creation of transgenic N. benthamiana plants expressing the "readthrough" isoform of human AChE (AChE-R) [11, 12]. This isoform is the monomeric and soluble product of one of the mRNA splice variants of the single human ACHE gene, which is up-regulated during exposure to anticholinesterase agents . The human enzyme was engineered to contain the endoplasmic reticulum (ER) retention signal KDEL (Fig. 1a). This modification, as well as codon optimization of the gene, enabled the recombinant protein to accumulate to high levels in leaves [10, 12]. We selected one of the highest expresser line, 2D, harboring (at least) four copies of the transgene and expressing AChE-RER at 0.3%–1% of total soluble protein (TSP) as the parental source of explants for the generation of hairy root cultures [11, 12].
Thirteen independent hairy root lines were generated by A. rhizogenes infection and screened for the presence of the transgene and for levels of recombinant protein, assayed by its enzymatic activity (Fig. 1b). These kanamycin-resistant clones displayed a wide distribution of recombinant protein accumulation, with the highest clone accumulating the transgenic product at 3.3% TSP while the lowest confirmed positive clones expressed at levels that were at least 100 fold lower. We observed a similarly wide distribution of recombinant protein accumulation levels in hairy-root clones derived from a single parental plant with the synaptic AChE isoform (data not shown).
The differences in the apparent expression levels of AChE-RER between the hairy root cultures and the parental plants can be explained by several, non-mutually exclusive explanations. For example it is reasonable to expect differences in the size of total soluble protein fraction in roots vs. leaves. It can also reflect a less restrained transgene expression under the pampered culture conditions as opposed to expression levels that can be expected of potted plants in the greenhouse. Regardless of the explanation, the higher % TSP observed in hairy roots presents itself as a bonus when purification is concerned.
Growth kinetics of AChE-RERhairy root cultures
WT hairy roots remained viable when stored at 4°C on solid medium (agar slants) and could be used to inoculate new suspension cultures for at least 6 months. However, and correlating well to its diminished growth characteristics, AChE-expressing hairy roots remained viable for only 15–20 weeks. Methods for longer-term clone banking are still being pursued.
Purification of AChE-RERfrom hairy root cultures
Hairy-root derived recombinant AChE-RER purification.
The purification results presented here, demonstrate the potential for further improvements in increasing the yield. A relative simple modification may be, for example, using a variant of the protein devoid of the ER retention signal, which should allow the protein to be secreted to the apoplast. It is yet to be seen if such approach would allow the ~70 kDa protein to be released into the medium (as opposed to being trapped within the plant cell wall and subsequent uptake and degradation) as the existing literature is ambivalent about the issue [21–23].
Pharmacokinetics of Hairy Root-Derived AChE-RER
Previously, we reported the creation of transgenic plants that accumulate recombinant human AChE-RER to commercially viable levels . Here we demonstrate that the enzyme can be efficiently produced in hairy root cultures derived from those transgenic plants, that it can be readily purified and that it is 'biosimilar', i.e. biochemically and functionally equivalent to its transgenic plant-derived counterpart with respect to substrate hydrolysis, OP binding and pharmacokinetics . It is anticipated, but yet to be demonstrated, that the hairy root enzyme can provide similar protection to OP challenged enzymes. Thus organ cultures can provide both the high level of expression achieved with transgenic plants, with the additional containment and uniformity coming from contained clonal propagation in well-defined culture medium and conditions.
This work was funded in part by the National Institutes of Health CounterACT Program through the National Institute of Neurological Disorders and Stroke under the U-54-NSO58183-01 award – a consortium grant awarded to USAMRICD and contracted to TSM under the research cooperative agreement number W81XWH-07-2-0023. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the federal USA government.
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