High-level expression of functional recombinant human butyrylcholinesterase in silkworm larvae by Bac-to-Bac system

doi:10.1016/j.cbi.2010.03.055

Chemico-Biological Interactions

Volume 187, Issues 1–3, 6 September 2010, Pages 101-105

https://doi.org/10.1016/j.cbi.2010.03.055 Get rights and content

Abstract

Butyrylcholinesterase (BChE: EC 3.1.1.8) serves as a natural scavenger for a variety of drugs, poisons, and organophosphorous compounds by hydrolyzing their ester bonds. Large scale production of recombinant human BChE (rhBChE) has been reported in transgenic goat. Here we demonstrate high-level expression of rhBChE with biological activity comparable to that of natural and recombinant enzymes, through the Bac-to-Bac baculovirus expression system in silkworm Bombyx mori larvae. We constructed the full-length hBChE cDNA into the plasmid pFastBac™. To monitor the level of expression, the cDNA coding for an orange fluorescent protein (OFP) was cloned downstream to the polyhedron (pH) promoter. Transfection was carried out by subcutaneous injection of 4–5th instar silkworm larvae. Approximately 4–7 days after infection, high-level expression of recombinant proteins was observed as indicated by the orange fluorescence of the larvae under blue light illumination. The hemolymph of the infected larvae was harvested, purified and assayed for BChE activity. The total units of BChE activity after purification were around 6.4 units per larvae. The K_m and V_max values of rhBChE were determined to be 17.7 μM and 2194 U/l hemolymph, respectively. By SDS-PAGE and Western analysis, the size of silkworm rhBChE was estimated to be 85 kDa. The results indicate that the silkworm larva is a good alternative system to produce bioactive rhBChE. Further optimization and modifications will be necessary for large-scale production of rhBChE. This should provide a rapid, low-cost, and high yield rhBChE for therapeutic applications.

Introduction

Butyrylcholinesterase (BChE: EC 3.1.1.8), an enzyme which is also known as serum cholinesterase or pseudocholinesterase, is able to hydrolyze many ester-containing drugs such as cocaine and succinylcholine [1], [2]. Consequently, BChE can be considered as an endogenous scavenger of anti-cholinesterase compounds. It detoxifies them before they reach AChE at physiologically important target sites [3]. BChE is also known to be a good scavenger of organophosphorous pesticides and chemical warfare nerve agents. For example, injections of purified BChE as pretreatment against nerve agent poisoning in mice, rats and monkeys increased their survival with a higher efficiency than the classical pretreatment with pyridostigmine [4], [5], [6]. Therefore, BChE could potentially be used for the detoxification of various drugs, poisons, and organophosphorous compounds. Native BChE has been purified in large quantity from human plasma [7]. Large amounts of recombinant BChE have been produced in transgenic goat [8] and in cell culture for structural determination [9]. It has also been reported that BChE was expressed in silkworm larvae using baculovirus infection with 23-fold higher than that in BmN cells [10].

Baculovirus expression system has become the method of choice for industrial expression of eukaryotic recombinant proteins. Traditionally, the recombinant baculovirus vector has been produced by homologous recombination at a low recombination frequency. The recently developed Bac-to-Bac expression system has quickly become a rapid and efficient method for recombinant baculovirus preparation [11]. It involves the site-specific transposition between a recombinant plasmid containing the target gene and a bacmid in bacteria. The colonies containing the recombinant bacmid were identified by white/blue selection based on the disruption of the lacZα gene. We report herein the high-level expression of recombinant human BChE (rhBChE) in the silkworm larvae using the Bac-to-Bac expression system.

Section snippets

Raising silkworms

Hybrid strain of silkworm larvae (commercial name: Qixiang Jiufu) was obtained from Guangdong Academy of Agricultural Sciences. Silkworm larvae were raised in a sterile environment on sterile artificial food at 23–25 °C with 70–90% humidity. After the fourth ecdysis, larvae were divided into groups for injection of recombinant bacmid DNA.

Construction of the expression cassette

The pFastBac™ DUAL plasmid was purchased from Invitrogen (San Diego, USA) which contains the expression cassettes driven by two strong promoters, p10 and pH.

High-level expression of rhBChE in silkworm larvae

Expression of rhBChE was detected on the 4th or 5th day post-injection. The highest expression level was achieved after 7–8 days post-injection. The silkworm larvae that received no injection of any recombinant bacmid DNA did not emit fluorescent light when exposed to blue light (Fig. 1B). The endogeneous BChE activity in these larvae was determined to be very low. In contrast, the silkworm larvae that received injections of rBacmid/BmNPV/(OFP) DNA (Fig. 1C) and rBacmid/BmNPV/(hBChE;OFP) DNA (

Discussion

In this study, we have successfully demonstrated a high-level expression of recombinant human butyrylcholinesterase in silkworm larvae by using the Bac-to-Bac system. This donor plasmid allowed the expression of two heterologous proteins simultaneously, therefore, a marker protein, such as the fluorescent proteins, was able to co-express with the target protein, providing us a convenient method to monitor the expression of target protein by directly visualizing the color of the silkworm.

Conflict of interest

The authors declare that they have no proprietary, financial, and other personal interest of any nature or kind in any product and company that would be construed as influencing the manuscript.

Acknowledgements

This work is partially supported by grants from the Research Grants Council of the Hong Kong Special Administrative Region, China (Project no. CUHK4311/03M and CUHK4571/05M) and GRF #474808 to DCW.

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High-level expression of human butyrylcholinesterase gene in Bombyx mori and biochemical–pharmacological characteristic study of its product
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Yeast-derived (Pichia Pastoris) rBChE also demonstrated poor stability and rapid degradation of up to 90% of the secreted protein in 48 h [43,44]. Expression of rBChE using a range of insect cells and larvae, have achieved yields up to 35 mg/L but proven useful only for crystallisation studies, with proteins lacking tetrameric structure and human-specific glycosylation [45–48]. It was reported that a 400 mg rBChE dose at a cost of $250 using either semi-continuous bioreactor production with rice cell suspension cultures or stable expression in tobacco could be achieved [24,49].
Organophosphorus nerve agents represent a serious chemical threat due to their ease of production and scale of impact. The recent use of the nerve agent Novichok has re-emphasised the need for broad-spectrum medical countermeasures (MCMs) to these agents. However, current MCMs are limited. Plasma derived human butyrylcholinesterase (huBChE) is a promising novel bioscavenger MCM strategy, but is prohibitively expensive to isolate from human plasma at scale. Efforts to produce recombinant huBChE (rBChE) in various protein expression platforms have failed to achieve key critical attributes of huBChE such as circulatory half-life. These proteins often lack critical features such as tetrameric structure and requisite post-translational modifications. This review evaluates previous attempts to generate rBChE and assesses recent advances in mammalian cell expression and protein engineering strategies that could be deployed to achieve the required half-life and yield for a viable rBChE MCM. This includes the addition of a proline-rich attachment domain, fusion proteins, post translational modifications, expression system selection and optimised downstream processes. Whilst challenges remain, a combinatorial application of these strategies demonstrates potential as a technically feasible approach to achieving a bioactive and cost effective bioscavenger MCM.
Proline 285 is integral for the reactivation of organophosphate-inhibited human butyrylcholinesterase by 2-PAM*
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The resultant cholinergic crisis, if left untreated, can lead to the development of generalized tonic-clonic seizures and death due to respiratory failure. Animals pretreated with AChE purified from fetal bovine serum [4] or BChE purified from human or horse serum or HuBChE produced in transgenic goats, tobacco plants, or insect larvae are protected from nerve agent toxicity [5–8]. These enzymes are bioscavengers that capture nerve agents before they reach their target and inhibit neurotransmission.
Human butyrylcholinesterase (HuBChE) is a stoichiometric bioscavenger that protects from the toxicity of nerve agents. Non-human primates are suitable models for toxicity studies that cannot be performed in humans. We evaluated the biochemical properties of native macaque (MaBChE) tetramers, compared to recombinant MaBChE monomers, PEGylated recombinant MaBChE tetramers and monomers, and native HuBChE tetramers. K_m and k_cat values for butyrylthiocholine were independent of subunit assembly status. The K_m for all forms of MaBChE was about 70 μM, compared to 13 μM for HuBChE. The k_cat was about 100,000 min⁻¹ for MaBChE and 30,000 min⁻¹ for HuBChE. The reversible inhibitor ethopropazine had similar K_i values of 0.05 μM for all MaBChE forms and HuBChE. The bimolecular rate constant, ki, for inhibition by diisopropylfluorophosphate (DFP), an analog of sarin, was 2.2 to 2.5 × 10⁷ M⁻¹ min⁻¹ for all MaBChE forms and for HuBChE. A major difference between MaBChE and HuBChE was the rate of reactivation by 2-PAM. The second order rate constant for reactivation of DFP-inhibited MaBChE by 2-PAM was 1.4 M⁻¹ min⁻¹, but was 380 fold faster for DFP-inhibited HuBChE (k_r 531 M⁻¹ min⁻¹). The acyl pocket of MaBChE has Leu285 in place of Pro285 in HuBChE. The reactivation rate of DFP-inhibited HuBChE mutant P285L by 2-PAM was reduced 5.8-fold (k_r 92 M⁻¹ min⁻¹) indicating that P285 determines whether 2-PAM binds in an orientation that favors release of diisopropylphosphate. DFP-inhibited MaBChE treated with 0.2 M 2-PAM recovered 10% of its original activity, whereas DFP-inhibited HuBChE recovered 80% activity. It was concluded that the biochemical properties of MaBChE are similar to those of HuBChE except for the reactivation of DFP-inhibited BChE.
Human umbilical cord perivascular cells: A novel source of the organophosphate antidote butyrylcholinesterase
2019, Chemico-Biological Interactions
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The benchmark BChE isoform for therapeutic use, exhibiting the longest half-life and optimal bioscavenging activity, is the optimally glycosylated tetrameric BChE complex [26–30]. A variety of eukaryotic recombinant platforms tested to date have lacked the innate capacity to produce the properly glycosylated tetrameric complex, resulting in a BChE product with a truncated half-life and sub-optimal bioscavenging functionality [31–40]. Bacterial platforms have proven even more challenging, since the recombinant BChE becomes sequestered in inclusion bodies [41,42].
Human butyrylcholinesterase (BChE) is a well-characterized bioscavenger with significant potential as a prophylactic or post-exposure treatment for organophosphate poisoning. Despite substantial efforts, BChE has proven technically challenging to produce in recombinant systems. Recombinant BChE tends to be insufficiently or incorrectly glycosylated, and consequently exhibits a truncated half-life, compromised activity, or is immunogenic. Thus, expired human plasma remains the only reliable source of the benchmark BChE tetramer, but production is costly and time intensive and presents possible blood-borne disease hazards. Here we report a human BChE production platform that produces functionally active, tetrameric BChE enzyme, without the addition of external factors such as polyproline peptides or chemical or gene modification required by other systems. Human umbilical cord perivascular cells (HUCPVCs) are a rich population of mesenchymal stromal cells (MSCs) derived from Wharton's jelly. We show that HUCPVCs naturally and stably secrete BChE during culture in xeno- and serum-free media, and can be gene-modified to increase BChE output. However, BChE secretion from HUCPVCs is limited by innate feedback mechanisms that can be interrupted by addition of miR 186 oligonucleotide mimics or by competitive inhibition of muscarinic cholinergic signalling receptors by addition of atropine. By contrast, adult bone marrow-derived mesenchymal stromal cells neither secrete measurable levels of BChE naturally, nor after gene modification. Further work is required to fully characterize and disable the intrinsic ceiling of HUCPVC-mediated BChE secretion to achieve commercially relevant enzyme output. However, HUCPVCs present a unique opportunity to produce both native and strategically engineered recombinant BChE enzyme in a human platform with the innate capacity to secrete the benchmark human plasma form.
General Overview
2014, Best Synthetic Methods: Organophosphorus (V) Chemistry
The element phosphorus; principal compounds of phosphorus; phosphorus compounds in living systems; a brief history of synthetic organophosphorus (OP) chemistry; nomenclature of OP compounds; phosphorus-containing pharmaceuticals; fire retardants and fire extinguishants; toxicology and medical treatment of poisoning by OP compounds; fluorogenic nerve agent probes; activity-based enzyme probes; and recommended safety procedures for handling OP compounds; are discussed.
Progress in the development of enzyme-based nerve agent bioscavengers
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Citation Excerpt :
Therefore, production of recombinant hBChE (rhBChE) has been sought for the last decade. rhBChE has been produced in different expression systems including CHO cells [32], drosophila cells [33], silkworm larvae [34], but economically viable large-scale production are currently limited to transgenic goats [35] and tobacco plants [31,36]. Transgenic goat milk rhBChE used to be considered as the most promising production system, but a compromised lactation performance due to endoplasmic reticulum stress put a halt to development [37,38].
Acetylcholinesterase is the physiological target for acute toxicity of nerve agents. Attempts to protect acetylcholinesterase from phosphylation by nerve agents, is currently achieved by reversible inhibitors that transiently mask the enzyme active site. This approach either protects only peripheral acetylcholinesterase or may cause side effects. Thus, an alternative strategy consists in scavenging nerve agents in the bloodstream before they can reach acetylcholinesterase. Pre- or post-exposure administration of bioscavengers, enzymes that neutralize and detoxify organophosphorus molecules, is one of the major developments of new medical counter-measures. These enzymes act either as stoichiometric or catalytic bioscavengers.
Human butyrylcholinesterase is the leading stoichiometric bioscavenger. Current efforts are devoted to its mass production with care to pharmacokinetic properties of the final product for extended lifetime. Development of specific reactivators of phosphylated butyrylcholinesterase, or variants with spontaneous reactivation activity is also envisioned for rapid in situ regeneration of the scavenger.
Human paraoxonase 1 is the leading catalytic bioscavenger under development. Research efforts focus on improving its catalytic efficiency toward the most toxic isomers of nerve agents, by means of directed evolution-based strategies. Human prolidase appears to be another promising human enzyme. Other non-human efficient enzymes like bacterial phosphotriesterases or squid diisopropylfluorophosphatase are also considered though their intrinsic immunogenic properties remain challenging for use in humans. Encapsulation, PEGylation and other modifications are possible solutions to address this problem as well as that of their limited lifetime.
Finally, gene therapy for in situ generation and delivery of bioscavengers is for the far future, but its proof of concept has been established.
Purification of recombinant human butyrylcholinesterase on Hupresin®
2018, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Affinity chromatography on procainamide-Sepharose has been an important step in the purification of butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) since its introduction in 1978. The procainamide affinity gel has limitations. In the present report a new affinity gel called Hupresin® was evaluated for its ability to purify truncated, recombinant human butyrylcholinesterase (rHuBChE) expressed in a stably transfected Chinese Hamster Ovary cell line. We present a detailed example of the purification of rHuBChE secreted into 3940 mL of serum-free culture medium. The starting material contained 13,163 units of BChE activity (20.9 mg). rHuBChE was purified to homogeneity in a single step by passage over 82 mL of Hupresin® eluted with 0.1 M tetramethylammonium bromide in 20 mM TrisCl pH 7.5. The fraction with the highest specific activity of 630 units/mg contained 11 mg of BChE. Hupresin® is superior to procainamide-Sepharose for purification of BChE, but is not suitable for purifying native AChE because Hupresin® binds AChE so tightly that AChE is not released with buffers, but is desorbed with denaturing solvents such as 50% acetonitrile or 1% trifluoroacetic acid. Procainamide-Sepharose will continue to be useful for purification of AChE.

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Chemico-Biological Interactions

Abstract

Introduction

Section snippets

Raising silkworms

Construction of the expression cassette

High-level expression of rhBChE in silkworm larvae

Discussion

Conflict of interest

Acknowledgements

References (15)

Therapeutic use of butyrylcholinesterase for cocaine intoxication

Toxicol. Appl. Pharmacol.

Genetic variants of human serum cholinesterase influence metabolism of the muscle relaxant succinylcholine

Pharmacol. Ther.

Human butyrylcholinesterase as a general prophylactic antidote for nerve agent toxicity. In vitro and in vivo quantitative characterization

Biochem. Pharmacol.

Butyrylcholinesterase and acetylcholinesterase prophylaxis against soman poisoning in mice

Biochem. Pharmacol.

Prophylaxis against soman inhalation toxicity in guinea pigs by pretreatment alone with human serum butyrylcholinesterase

Toxicol. Sci.

High-level expression of human butyrylcholinesterase gene in Bombyx mori and biochemical–pharmacological characteristic study of its product

Biochem. Pharmacol.

Anti-acetylcholinesterase activities of traditional Chinese medicine for treating Alzheimer's disease

Chem. Biol. Interact.

Cited by (18)

Strategies for developing a recombinant butyrylcholinesterase medical countermeasure for Organophosphorus poisoning

Proline 285 is integral for the reactivation of organophosphate-inhibited human butyrylcholinesterase by 2-PAM*

Human umbilical cord perivascular cells: A novel source of the organophosphate antidote butyrylcholinesterase

General Overview

Progress in the development of enzyme-based nerve agent bioscavengers

Purification of recombinant human butyrylcholinesterase on Hupresin®