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

Chemico-Biological Interactions 187 (2010) 101–105

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High-level expression of functional recombinant human butyrylcholinesterase in silkworm larvae by Bac-to-Bac system Shuo Li a , Denis Tsz Ming Ip a , Huang Quan Lin a , Jian Mei Liu b , Yun Gen Miao b , Li Jian Ke a , David Chi Cheong Wan a,∗ a b

School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China College of Animal Sciences, Zhejiang University, Hangzhou, China

a r t i c l e

i n f o

Article history: Available online 24 April 2010 Keywords: Butyrylcholinesterase Bac-to-Bac system Silkworm (Bombyx mori L.) Purification

a b s t r a c t 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 pFastBacTM . 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 Km and Vmax values of rhBChE were determined to be 17.7 ␮M 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. © 2010 Elsevier Ireland Ltd. All rights reserved.

1. 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 [4–6]. Therefore, BChE could potentially be used for the detoxification of various drugs, poi-

∗ Corresponding author at: Rm 178, Science Center, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong SAR, China. Tel.: +852 2609 6252; fax: +852 2603 7246. E-mail address: [email protected] (D.C.C. Wan). 0009-2797/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2010.03.055

sons, 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 sitespecific 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 lacZ␣ gene. We report herein the high-level expression of recombinant human BChE (rhBChE) in the silkworm larvae using the Bac-to-Bac expression system.

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Fig. 1. (A) The schematic diagram of recombinant rBacmid/BmNPV/(hBChE;OFP) DNA. The recombinant bacmid DNA contained an expression cassette flanked by the left and right arms of Tn7, and inside the cassette, hBChE and OFP were inserted into the multiple cloning sites downstream of the p10 and pH promoters, respectively. (B) Blank control: silkworm larvae not injected with recombinant bacmid. No OFP and rhBChE was expressed. (C) OFP control: silkworm larvae injected with BmNPV(OFP) DNA. Only OFP was expressed. (D) Silkworm larvae injected with BmNPV/(hBChE;OFP) DNA. Both rhBChE and OFP were expressed in its body. (The left panels were captured under daylight, the right ones were captured under a blue-light illuminator.)

2. Materials and methods 2.1. 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. 2.2. Construction of the expression cassette The pFastBacTM DUAL plasmid was purchased from Invitrogen (San Diego, USA) which contains the expression cassettes driven by two strong promoters, p10 and pH. The full length of native hBChE cDNA including the signal sequence (1.8 kb) was cloned into the Xho I to Kpn I sites under the control of p10 promoter. The mutated gene of orange fluorescent protein OFP that expresses a dimeric form of OFP [12] was sub-cloned into the Not I to Hind III sites under the control of pH promoter. 2.3. Construction of the recombinant bacmid baculovirus The recombinant baculovirus harboring the human BChE gene was generated by site-specific transposition in DH10BacTM Escherichia coli. The recombinant bacmid baculovirus was designated as rBacmid/BmNPV/(hBChE:OFP) (Fig. 1A). The E. coli DH10Bac

containing recombinant bacmid DNA was propagated on LB medium containing antibiotics (50 ␮g/ml kanamycin, 7 ␮g/ml gentamicin, and 10 ␮g/ml tetracycline), 100 ␮g/ml X-gal and 40 ␮g/ml IPTG. The white colonies which contain the recombinant bacmid were picked for further confirmation by PCR. 2.4. Expression of the rhBChE in silkworm larvae The recombinant bacmid DNA was transfected into silkworm larvae with the help of Cellfectin (Invitrogen, USA). The final concentration of the recombinant bacmid DNA in the mixture was 0.02 ␮g/␮l. An aliquot of 10 ␮l was injected into the dorsal side of each silkworm larvae with a syringe. The silkworm larvae on the 7th day after injection were examined under blue light with an average wavelength of 475 nm to observe the presence of orange fluorescence. The hemolymph were collected and centrifuged at 500 × g for 5 min to remove insoluble materials. The recombinant baculovirus was obtained from the clarified supernatant. 2.5. Oral infection of silkworm larvae The silkworm hemolymph harvested from the first batch of infected larvae was orally administered to the second batches of silkworms by oral infection. In order to achieve a higher infection rate, the silkworms were chilled at 4 ◦ C. The duration of chilling treatment varied from 4 to 24 h. After awakening from the temporary diapause, the silkworms were fed

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Fig. 2. Two-step purification profile of rhBChE (A) elution profile of rhBChE from procainamide-Sepharose 2B affinity column. A total volume of 2 ml hemolymph was subjected to the column. Enzyme was eluted with 0.2 mol/l procainamide hydrochloride in PBS (10 mmol/l, pH 7.4, containing 1 mmol/l EDTA). Fractions (0.2 ml) were collected and assayed for activity. (B) SDS-PAGE (left one) and western blot (right one) analysis after the first step of purification. Lane 1 was the hemolymph without purification; Lane 2 was the fraction collected from the affinity column. (C) HPLC profile of rhBChE. 20 ␮l of solution collected from the peak of the affinity column was injected into HPLC. The first peak having retention time of 5.72 min with absorbance at 280 nm was confirmed to be rhBChE by Ellman Assay. (D) The target protein appeared as a sharp single band with the size of 85 kDa on SDS-PAGE after the second step of purification.

artificial food coated or impregnated with the recombinant baculovirus.

affinity column with 0.2 mol/l procainamide hydrochloride in PBS (10 mmol/l, pH 7.4, containing 1 mmol/l EDTA).

2.6. BChE activity assay

2.8. SDS-PAGE and western blot analysis of rhBChE

The enzyme activity was determined with the substrate butyrylthiocholine (12.5 mM; Sigma, B3253-5G) in a total volume of 200 ␮l sodium phosphate buffer (100 mM, pH 7.4) at 37 ◦ C according to the Ellman method as modified previously in our lab [13].

The enzyme sample was applied to the SDS-PAGE with 12% acrylamide in separating gel and 5% in stacking gel. The protein bands were stained with Coomassie blue. Western blot was also performed. The anti-BChE polyclonal goat antibody (Santa Cruz Biotechnology Inc., sc-46803) was added into 1× TBS-T buffer and incubated with the membrane on which the rhBChE had been transferred, so the primary antibody would bind onto the rhBChE. Then the AP conjugated secondary antibody (ZYMED, 81-1622) was added and bound onto the primary antibody. After adding substrate and colorimetric reagent, rhBChE on the membrane was visualized.

2.7. Purification of rhBChE from hemolymph of silkworm larvae The purification was carried out in a two-step process. rhBChE was first purified by procainamide-Sepharose 4B affinity chromatography, then followed by high performance gel filtration (Phenomenex, BioSep-SEC-S 2000). Sepharose 4B affinity gel was prepared according to Grunwald et al. [14]. Procainamide (Sigma, P9391-25G) (0.1 mmol/ml of gel) was coupled to the activated CHSepharose 4B (GE healthcare, 17-0490-01) with a 0.1 M solution of 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide hydrochloride at pH 4.5 by titrating with 1 M HCl. Then rhBChE was eluted from the

2.9. Kinetic analysis The rhBChE activity was determined according to the method of Ellman et al. with minor modifications. Butyrylthiocholine (BSCh) and acetylthiocholine (ASCh) (Sigma, A-5751) were used as substrates to examine the specificity of rhBChE towards them,

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respectively. Various substrate concentrations ranging from 0.0024 to 30 mM reacted with the enzyme in a total volume of 200 ␮l of sodium phosphate buffer (100 mM, pH 7.4) at 37 ◦ C, then 10 ␮l of 10 mM DTNB was added and the absorbance was measured at 412 nm. The increase in absorbance was measured over time interval of 3 min. The data were analyzed by the equation reported in Radic et al. [15] and the parameters of Km , b, Kss and Vmax were determined using the curve fitting algorithm (KaleidaGraph). 3. Results 3.1. High-level expression of rhBChE in silkworm larvae Expression of rhBChE was detected on the 4th or 5th day postinjection. 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 (Fig. 1D) emitted bright orange fluorescent light upon exposure to blue light, which indicated that the target protein had been expressed successfully in their body. Enzymatic activity of rhBChE (U/ml) was defined as the micromoles butyrylthiocholine hydrolyzed per minute per ml. Approximately 0.5–1 ml hemolymph can be obtained from each 5th instar larva. Around 6.4 units of purified rhBChE can be obtained from each larva. The Km and Vmax values of rhBChE were determined to be 17.7 ␮M and 2194 U/l, respectively. The baculovirus was obtained from the clarified supernatant of the hemolymph which could be stored as seed to infect other silkworm larvae by direct dermal or oral infection. 3.2. Oral infection of silkworm larvae The chilling treatment was carried out at 4 ◦ C for different duration (4 h, 12 h, 18 h, 24 h) in order to examine the optimal pre-chilling time for silkworm larvae baculovirus infection. After chilling, the silkworms were brought back to the room temperature. They were fed with artificial food coated or impregnated with the recombinant virus rBacmid/BmNPV/ (hBChE;OFP) obtained from diluted infected hemolymph. It was found that there was a correlation between the time of the chilling treatment and its infection rate; the infection rate was highest (>80%) when chilling was for 24 h. In order to investigate the minimum dose of virus that could lead to the infection of the silkworm larvae, the hemolymph containing the recombinant virus was diluted in a 10-fold manner (10−1 , 10−2 , 10−3 ). It was found that 1000-fold dilution of the infected hemolymph was still capable of infecting the silkworms by oral consumption. 3.3. Purification of rhBChE from silkworm larvae hemolymph BChE from hemolymph was purified by ProcainamideSepharose 4B affinity column chromatography. The activity of rhBChE in the different fractions collected from the affinity column is shown in Fig. 2A, about 70% of rhBChE activity was recovered from this step. The fractions in the peak were collected for SDSPAGE and western analysis (Fig. 2B). The target protein band was located at 85 kDa, which corresponded to the size of the hBChE monomer. In order to obtain a highly purified rhBChE, the affinity purified enzyme was subjected to high performance gel filtration (Fig. 2C). A single peak at the retention time of 5.72 min was

Fig. 3. Kinetic studies of rhBChE towards different substrates. Increasing concentration of substrate was added to the assay mixture and enzyme activity was measured according to the method in the text. The data were analyzed by the equation reported in Radic et al. and the parameters of Km , b, Kss and Vmax were determined using the curve fitting algorithm (KaleidaGraph). Filled squares () present BSCh, filled circles (䊉) present ASCh. Km , b and Kss values of rhBChE towards BSCh (Km = 17.7 ␮M, b = 3.3, Kss = 1.6 mM, Vmax = 2194 U/l) and ASCh (Km = 46.6 ␮M, b = 3.9, Kss = 12 mM, Vmax = 1810 U/l) were determined by curve fitting using the equation: ((1 + b[S]/Kss )/(1 + [S]/Kss ))(Vmax /(1 + Km /[S])).

recovered which has the molecular size of 85 kDa on SDS-PAGE (Fig. 2D). 3.4. Kinetic studies of rhBChE activity The purified enzyme from gel filtration was used for kinetic studies. Butyrylthiocholine (BSCh) and acetylthiocholine (ASCh) were used as substrates. The activity of rhBChE with BSCh was greater than with ASCh (Fig. 3). The Km and Vmax values of rhBChE towards BSCh were 17.7 ␮M and 2194 U/l, which were much higher than that of ASCh (46.6 ␮M and 1810 U/l). 4. 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. Besides, this Dual vector can also be used to study protein–protein interaction by expressing two foreign proteins together. The yield of enzyme activity in the hemolymph of silkworm larvae was determined to be 2194 U/l, which was comparable to that of the hemolymph of silkworm larvae previously reported by Wei et al. [10]. The Bacto-Bac expression system reported here is also much less tedious to manipulate which took weeks instead of months to obtain the recombinant baculovirus. We also demonstrate that the recombinant baculovirus is able to re-infect the larvae by oral route without tedious subcutaneous injection. The infection efficiency was optimal when the larvae were pre-chilled. It was the most economical means to produce large amounts of recombinant proteins which is amenable for industrial production of recombinant proteins. During the course of this work, we became aware of a US company Chesapeake PERL (www.c-perl.com) at which they used a proprietary baculovirus expression system to infect the larvae of cabbage looper (Trichoplu-

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sia ni) for expression of rhBChE. The silkworm larvae expression system described here represents an alternative method which is more economical as the silkworm farming industry is well established in China. Therefore, silkworm larvae are more suitable for industrial production of recombinant proteins especially in China. In our study, we showed that the molecular size of rhBChE is 85kDa, which is very similar to the commercial native hBChE. Wei et al. [10] reported the expression of rBChE with the molecular weight of 71 kDa by using baculovirus expression system in silkworm larvae. The difference in molecular size may be due to the different silkworm strains used which lead to the different degree of glycosylation. In conclusion, we have demonstrated a high-level expression here the expression of functional recombinant human butyrylcholinesterase in silkworm larvae by the Bac-to-Bac expression system. Further investigation on the large-scale expression of rhBChE could provide us a rapid, low-cost, and high yield method to produce rhBChE and facilitate the development of its potential therapeutic applications. 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. References [1] C.E. Mattes, T.J. Lynch, A. Singh, R.M. Bradley, P.A. Kellaris, R.O. Brady, K.L. Dretchen, Therapeutic use of butyrylcholinesterase for cocaine intoxication, Toxicol. Appl. Pharmacol. 145 (2) (1997) 372–380.

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[2] O. Lockridge, Genetic variants of human serum cholinesterase influence metabolism of the muscle relaxant succinylcholine, Pharmacol. Ther. 47 (1) (1990) 35–60. [3] J. Viby-Mogensen, Succinylcholine neuromuscular blockade in subjects heterozygous for abnormal plasma cholinesterase, Anesthesiology 55 (3) (1981) 231–235. [4] L. Raveh, J. Grunwald, D. Marcus, Y. Papier, E. Cohen, Y. Ashani, Human butyrylcholinesterase as a general prophylactic antidote for nerve agent toxicity. In vitro and in vivo quantitative characterization, Biochem. Pharmacol. 45 (12) (1993) 2465–2474. [5] Y. Ashani, S. Shapira, D. Levy, A.D. Wolfe, B.P. Doctor, L. Raveh, Butyrylcholinesterase and acetylcholinesterase prophylaxis against soman poisoning in mice, Biochem. Pharmacol. 41 (1) (1991) 37–41. [6] N. Allon, L. Raveh, E. Gilat, E. Cohen, J. Grunwald, Y. Ashani, Prophylaxis against soman inhalation toxicity in guinea pigs by pretreatment alone with human serum butyrylcholinesterase, Toxicol. Sci. 43 (2) (1998) 121–128. [7] O. Lockridge, L.M. Schopfer, G. Winger, J.H. Woods, Large scale purification of butyrylcholinesterase from human plasma suitable for injection into monkeys; a potential new therapeutic for protection against cocaine and nerve agent toxicity, J. Med. Chem. Biol. Radiol. Def. 3 (2005) nihms5095. [8] E. Podoly, T. Bruck, S. Diamant, N. Melamed-Book, A. Weiss, Y. Huang, O. Livnah, S. Langermann, H. Wilgus, H. Soreq, Human recombinant butyrylcholinesterase purified from the milk of transgenic goats interacts with beta-amyloid fibrils and suppresses their formation in vitro, Neurodegener. Dis. 5 (3/4) (2008) 232–236. [9] M.N. Ngamelue, K. Homma, O. Lockridge, O.A. Asojo, Crystallization and Xray structure of full-length recombinant human butyrylcholinesterase, Acta Crystallogr. F: Struct. Biol. Cryst. Commun. 63 (Pt 9) (2007) 723–727. [10] W.L. Wei, J.C. Qin, M.J. Sun, High-level expression of human butyrylcholinesterase gene in Bombyx mori and biochemical–pharmacological characteristic study of its product, Biochem. Pharmacol. 60 (1) (2000) 121–126. [11] Y. Miao, Y. Zhang, K. Nakagaki, T. Zhao, A. Zhao, Y. Meng, M. Nakagaki, E.Y. Park, K. Maenaka, Expression of spider flagelliform silk protein in Bombyx mori cell line by a novel Bac-to-Bac/BmNPV baculovirus expression system, Appl. Microbiol. Biotechnol. 71 (2) (2006) 192–199. [12] D.T. Ip, K.B. Wong, D.C. Wan, Characterization of novel orange fluorescent protein cloned from cnidarian tube anemone Cerianthus sp., Mar. Biotechnol. (NY) 9 (4) (2007) 469–478. [13] H.Q. Lin, M.T. Ho, L.S. Lau, K.K. Wong, P.C. Shaw, D.C. Wan, Antiacetylcholinesterase activities of traditional Chinese medicine for treating Alzheimer’s disease, Chem. Biol. Interact. 175 (1–3) (2008) 352–354. [14] J. Grunwald, D. Marcus, Y. Papier, L. Raveh, Z. Pittel, Y. Ashani, Large-scale purification and long-term stability of human butyrylcholinesterase: a potential bioscavenger drug, J. Biochem. Biophys. Methods 34 (2) (1997) 123–135. [15] Z. Radic, N.A. Pickering, D.C. Vellom, S. Camp, P. Taylor, Three distinct domains in the cholinesterase molecule confer selectivity for acetyl- and butyrylcholinesterase inhibitors, Biochemistry 32 (45) (1993) 12074–12084.