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Expression, purification and characterization of the chitinolytic β-N-acetyl-d -hexosaminidase from the insect Ostrinia furnacalis
Protein Expression and Purification 68 (2009) 99–103
Contents lists available at ScienceDirect
Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep
Expression, purification and characterization of the chitinolytic b-N-acetyl-D-hexosaminidase from the insect Ostrinia furnacalis Tian Liu a,1, Fengyi Liu a,1, Qing Yang a,b,*, Jun Yang a a b
Department of Bioscience and Biotechnology, Dalian University of Technology, Dalian 116024, China State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China
a r t i c l e
i n f o
Article history: Received 8 May 2009 and in revised form 3 June 2009 Available online 9 June 2009 Keywords: b-N-acetyl-D-hexosaminidase Chitin degradation Expression Insect
a b s t r a c t Insect b-N-acetyl-D-hexosaminidases are of particular interest due to their multiple physiological roles in many life processes. Chitinolytic b-N-acetyl-D-hexosaminidases, which function only in chitin degradation in insects, have long been regarded as species-specific target potentials in developing environmental friendly pesticides. Here the chitinolytic b-N-acetyl-D-hexosaminidase from the insect Ostrinia furnacalis was cloned and expressed in the yeast strain, Pichia pastoris, to meet the demands of biochemical studies and drug development. Enzymatic assay as well as Western blot confirmed that the high-level expression could be achieved after the induction of methanol for 120 h. Through the sequential combination of ammonium sulfate precipitation, metal chelating chromatography as well as anion exchange chromatography, 7.7 mg of the recombinant OfHex1 with high purity was obtained from 1 liter of culture supernatant. The recombinant OfHex1, characterized as a homodimer with molecular weight of 130 kDa, exhibited the same enzymatic activities as its native form, which could efficiently degrade the chitooligosaccharide substrate (GlcNAc)2 and release 4-methylumbelliferone (4MU) from substrates, 4MU-b-GlcNAc and 4MU-b-GalNAc. This work provides a low-costing and high-efficient purification procedure for the preparation of insect b-N-acetyl-D-hexosaminidases. Ó 2009 Elsevier Inc. All rights reserved.
Introduction Chitin biodegradation exists in chitin-containing organisms such as fungi, crustaceans and insects, whose main structural component is chitin, and in some bacteria which utilize chitin as carbon and nitrogen sources. Two enzymes play key roles in this process: chitinase (EC 3.2.1.14) firstly catalyzes the degradation of chitin into chitooligosaccharides, and b-N-acetyl-D-hexosaminidase (EC 3.2.1.52) hydrolyzes chitooligosaccharides into N-acetyl2 D-glucosamine (GlcNAc) [1]. The ratio of these two enzymes regulates the efficiency of chitin degradation and thus affects remodeling of chitin components of insects and fungi [2]. In this regard, these enzymes could be specific target potentials in the development of environmental friendly pesticides [3,4]. On the other hand, since chitin is the second most abundant but underutilized biomass in nature, the degradation of chitin by chitinolytic enzymes is of biotechnological importance [5].
* Corresponding author. Address: Department of Bioscience and Biotechnology, Dalian University of Technology, No. 2 Linggong Road, Dalian 116024, China. Fax: +86 0411 84707245. E-mail address: [email protected] (Q. Yang). 1 These authors contributed equally to this work. 2 Abbreviations used: 4MU, 4-methylumbelliferone; GlcNAc, N-acetyl-D-glucosamine; GST, glutathione-S-transferase; TBS, tris buffer saline. 1046-5928/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2009.06.004
We previously reported an insect b-N-acetyl-D-hexosaminidase from the Asian corn borer, Ostrinia furnacalis (Guenée), designated as OfHex1 [6]. Unlike other b-N-acetyl-D-hexosaminidases which function in N-glycan modification [7–9], glycoconjugate degradation [10,11] or sperm–egg recognition [12], OfHex1 was identified as the first insect b-N-acetyl-D-hexosaminidase whose physiological role was only involved in chitin degradation. Therefore, the preparation of this enzyme is of significance in the view of biochemical studies and biotechnology. Three reports associated with the recombinant expression and purification of insect b-N-acetyl-D-hexosaminidases have been revealed using insect cell lines as expression system, in which the b-N-acetyl-D-hexosaminidase activity of the host cell could not be ruled out. The glutathione-S-transferase (GST) tagged Sf-FDL from Spodoptera frugiperda and Dm-FDL from Drosophila melanogaster were expressed in Sf9 cells and purified by GST affinity chromatography [9]. It is the only example of successful expression and purification of recombinant insect b-N-acetyl-D-hexosaminidases. The SfHex, SfGlcNAcase-1 and SfGlcNAcase-3 from S. frugiperda were expressed in Sf9 cells but failed to be purified [10,13]. Since yeast is the low-cost expression system for producing large quantities of eukaryotic proteins, the recombinant expression of insect b-N-acetyl-D-hexosaminidases in yeast was tried. FDL, HEXO1 and HEXO2 from D. melanogaster were cloned into the expression vector, pPICZaA, and expressed in Pichia pastoris GS115 without purification
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T. Liu et al. / Protein Expression and Purification 68 (2009) 99–103
[8]. BmGlcNAcase1 and BmGlcNAcase2 from Bombyx mori were cloned into the expression vectors, pPICZaB and pPICZaC, respectively, and expressed in P. pastoris GS115, but purification attempts were still unsuccessful [8,11]. Herein we report the insect b-N-acetyl-D-hexosaminidase, OfHex1, was cloned into the expression vector pPIC9 instead of pPICZa series and expressed in the yeast strain, P. pastoris GS115. Through two chromatographic separation steps, the recombinant OfHex1 with high purity was obtained. To our knowledge, this is the first example of successful expression and purification of insect b-N-acetyl-D-hexosaminidase in yeast system. Partial enzymatic characterization was also performed.
Materials and methods Materials Restrict enzymes were purchased from NEB (Ipswich, MA). p-Nitrophenyl-N-acetyl-b-D-glucosaminide, 4-methylumbelliferyl4-methylumbelliferyl-N-acetyl-b-D-glucosb-D-glucopyranoside, aminide, 4-methylumbelliferyl-N-acetyl-b-D-galactosaminide and (GlcNAc)2 were purchased from Sigma–Aldrich (St. Louis, MO). 4Methylumbelliferyl-6-sulfo-N-acetyl-b-D-glucosaminide, HisTagTM monoclonal antibody and rabbit anti mouse peroxidase-conjugated secondary antibody were purchased from Merck (San Diego, CA). (GlcNAcb1,2Mana1,6)(GlcNAcb1,2Mana1,3)Manb1,4GlcNAcb1,4 GlcNAc-PA was purchased from Takara, (Dalian, China). All the other chemicals were purchased from commercial sources with highest purity.
Enzyme activity and protein concentration assay The enzyme activity was measured using p-nitrophenyl-N-acetyl-b-D-glucosaminide (pNP-b-GlcNAc) as substrate [6]. Briefly, the reaction was stopped by adding an equal volume of 0.5 M Na2CO3 and absorbance at 405 nm was monitored by microplate reader (TECAN, Männedorf, Switzerland). The protein concentration was assayed by the method of Bradford [14]. Purification of the recombinant OfHex1 All the column chromatographies were performed by using ÄKTA Purifier system (GE Healthcare, Uppsala, Sweden). In the first step, solid ammonium sulfate was added to OfHex1-expressed supernatant to 65% saturation. After incubation at 4 °C for 1 h, the medium supernatant was centrifuged at 8000g for 30 min. Then the pellet was dissolved in buffer A (20 mM sodium phosphate (pH 7.4), containing 0.1 M NaCl) and centrifuged at 12,000g for 10 min. The supernatant was loaded onto an IMAC Sepharose High Performance column (20 ml, GE Healthcare, Uppsala, Sweden) pre-equilibrated with buffer A. The column was first washed with buffer A containing 75 mM imidazole and the 6His tagged OfHex1 was eluted with buffer A containing 150 mM imidazole. Then the sample was desalted by a HiTrap desalting column (53 ml, GE Healthcare, Uppsala, Sweden) with buffer B (20 mM bis–Tris, pH 6.5) and loaded onto a Q Sepharose High Performance column (10 ml, GE Healthcare, Uppsala, Sweden) pre-equilibrated with buffer B. After washing, the column was eluted with a linear gradient of NaCl from 75 to 275 mM in buffer B. Fractions (4 ml each) were collected and enzymatic activities were assayed. The purity was analyzed by SDS–PAGE.
Construction of expression plasmids SDS–PAGE and gel filtration chromatography The vector CTB557-2-1 carrying the gene encoding OfHex1 was stored in our lab. Based on the cDNA sequence of OfHex1 (GenBank Accession No. DQ887769), two primers, 50 -GCTTACGTAGAATTC GAGGACGTAGTATGGCGCTGGT-30 and 50 -TTAATTCGCGGCCGC TTA ATGATGATGATGATGATGCGAGTAACAGTACCCCTC-30 were synthesized to amplify gene encoding the mature OfHex1 by PCR from CTB557-2-1. Underlined bases correspond to restriction enzymes sequences of EcoRI/NotI, respectively, and bases in bold correspond to the sequence encoding 6His tag at C-terminal. The resulting PCR products were digested with EcoRI/NotI and then cloned into EcoRI/NotI cut pPIC9 vector (Invitrogen, Carlsbad, CA) to generate the expression plasmid named pPIC9-OfHex1.
SDS–PAGE was carried out as described by Laemmli [15], using 10% (w/v) polyacrylamide resolving gel and 4% (w/v) polyacrylamide stacking gel. After electrophoresis, proteins in gel were visualized with Coomassie Brilliant Blue. For analytical gel filtration, 50 ll of purified OfHex1 was applied to Superdex 200 10/30 GL column (GE Healthcare, Uppsala, USA) pre-equilibrated with 20 mM of sodium phosphate (pH 6.8) containing 150 mM NaCl. The column was eluted at a flow rate of 0.3 ml/min. The molecular weight of the recombinant OfHex1 was determined as described before [6]. Western blot
Transformation and expression of the recombinant OfHex1 The expression vector plasmid (pPIC9-OfHex1) was linearized using PmeI and electroporated into the P. pastoris GS115 cells. The selection of His+ and Mut+ P. pastoris transformant was performed according to the manufacturer’s instructions. Positive transformants were cultured in 2 ml of BMGY broth (2% peptone, 1% yeast extract, 1% glycerol, 1.34% yeast nitrogen base and 0.1 M potassium phosphate (pH 6.0)) in tubes for 24 h and then induced for 48 h by the addition of 1% of methanol at every 12 h intervals. The transformant with the highest enzyme activities in the culture supernatant was selected. The selected transformant was precultured in 100 ml of BMGY broth and then transferred to 1 L of BMMY broth (2% peptone, 1% yeast extract, 1% methanol, 1.34% yeast nitrogen base and 0.1 M potassium phosphate (pH 6.0)). The cultures were incubated at 29 °C with continuous shaking and methanol (1% of the total volume) was re-added every 24 h. After 120 h fermentation, the supernatant was collected by centrifugation at 8000g for 30 min.
The proteins were separated by 10% SDS–PAGE and transferred onto a polyvinylidene fluoride membrane. The membrane was blocked with 5% milk in Tris buffer saline (TBS) for 1 h and then incubated with HisTagTM monoclonal antibody (Merck, San Diego, CA) at a dilution of 1:1000 in 3% milk in TBS overnight at 4 °C. After washing, the membrane was incubated with a rabbit anti mouse peroxidase-conjugated secondary antibody (Merck, San Diego, CA) at a dilution of 1:7500 in 3% milk in TBS for 1 h. The blot was developed with 3,3-diaminobenzidine tetrahydrochloride solution. Characterization of the recombinant OfHex1 The determination of the optimal pH was performed in 100 mM Britton-Robinson’s wide range buffer from pH 3 to pH 11 with one pH scale interval using pNP-b-GlcNAc as substrate. For the determination of the optimal reaction temperature, the reaction mixtures were incubated at various temperatures from 10 °C to 60 °C with 10 °C interval.
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To characterize the substrate spectrum of the recombinant OfHex1, enzymatic activities were measured using different kinds of substrates. First, the 4-methylumbelliferone (4MU)-linked substrates including 4-methylumbelliferyl-b-D-glucopyranoside (4MU-b-Glc), 4-methylumbelliferyl-N-acetyl-b-D-glucosaminide (4MU-b-GlcNAc), 4-methylumbelliferyl-N-acetyl-b-D-galactosaminide (4MU-b-GalNAc) and 4-methylumbelliferyl-6-sulfo-N-acetylb-D-glucosaminide (4MU-b-GlcNAc-6-SO4) were applied. The reaction sample consisted of 175 ll of 20 mM bis–Tris buffer (pH 6.5), 20 ll of 1 mM 4MU-linked substrates and 5 ll of enzyme. After incubation at 25 °C for 5 min, 1 ml of 0.5 M Na2CO3 was added to the reaction sample. The released 4MU was monitored by F-4500 spectrofluorimeter (HITACHI, Tokyo, Japan) with excitation wavelength of 360 nm and emission wavelength of 450 nm. Second, the chitooligosaccharide substrate (GlcNAc)2 and the pyridylaminated N-glycan substrate (GlcNAcb1,2Mana1,6)(GlcNAcb1,2Mana1,3)Manb1,4GlcNAcb1,4GlcNAc-PA (GnGn-PA) were applied. The hydrolytic efficiencies of (GlcNAc)2 and GnGn-PA were measured by HPLC using TSKgel Amide-80 column (4.6 250 mm, Tosoh, Tokyo, Japan) and a Hypersil ODS2 column (4.6 250 mm, Thermo, Waltham, USA), respectively [6].
Results Cloning and expression of the recombinant OfHex1 The gene encoding mature OfHex1 was inserted into the yeast expression vector pPIC9. The a-factor signal sequence carried by the vector was used to promote secretory expression. A 6His tag was added to the C-terminal to facilitate purification. Correct insertion and reading frame of OfHex1 were confirmed by DNA sequencing. Then the resulting pPIC9-OfHex1 vector was linearized and electroporated into P. pastoris GS115 cells. The positive transformants were obtained by screening His+/Mut+ plates. The His+/Mut+ P. pastoris transformant was cultured and OfHex1 expression was induced by adding 1% methanol. OfHex1 expression was monitored every 12 h by measuring both b-N-acetyl-Dhexosaminidase activity and protein concentration in culture supernatant. The specific activity in culture supernatant was determined and used to determine the optimal induction time. As shown in Fig. 1, the highest specific activity with 2.06 U/mg of pro-
Fig. 1. Total activities and specific activities in the culture supernatant at different time of induction. The black squares and white squares represent total activities and specific activities in 1 L of culture supernatant. The enzyme activity was determined by using the substrate, pNP-b-GlcNAc, and measuring the liberation of pNP at 405 nm.
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tein was observed after the induction for 120 h, when 362 U of total activity were obtained in 1 L of culture supernatant. Purification of the recombinant OfHex1 The recombinant OfHex1 was purified to homogeneity from the culture supernatant by a sequential combination of ammonium sulfate precipitation, metal chelating as well as anion exchange chromatographies (Fig. 2A). Through ammonium sulfate precipitation, the sample was not only concentrated from 1 L to 20 ml but also purified by 2.16 folds (Fig. 2A, lane 1). The first-step column chromatography, Ni2+-chelating affinity chromatography, contributed to 5-fold purification efficiency (Table 1). By washing the column with buffer containing 75 mM of imidazole before eluting the target protein, most of the contaminating proteins were removed (Fig. 2A, lane 2). Then the residual impurities were removed by anion exchange chromatography (Fig. 2A, lane 3). About 7.7 mg of purified recombinant OfHex1 was obtained from 1 L of culture supernatant. The total purification efficiency was 11 folds, which was summarized in Table 1. The specific activity of the purified recombinant OfHex1 (23.85 U/mg) was similar to specific activity of the native OfHex1 (24.96 U/mg) [6]. The purified protein was resolved as a single band with molecular weight of 69 kDa by SDS–PAGE and verified by Western blot using anti-His tag antibody (Fig. 2A, lanes 3 and 5). The recombinant OfHex1 was also resolved as a single sharp peak by analytical gel filtration chromatography (Fig. 2B), whose molecular weight was determined to be 130 kDa by standard curve. Thus, the recombinant OfHex1 was in the same form of homodimer as the native OfHex1 [6].
Fig. 2. Purification of the recombinant OfHex1. (A) Lanes 1–3, SDS–PAGE analysis of purification efficiency of the recombinant OfHex1. Lane 1, sample after ammonium sulfate precipitation; lane 2, sample after metal chelate chromatography on IMAC Sepharose HP column; lane 3, the purified recombinant OfHex1 after anion exchange chromatography on Q Sepharose HP column. Lanes 4 and 5, Western blot (WB) of the recombinant OfHex1 by using HisTagTM monoclonal antibody. Lane 4, culture supernatant from cells harboring pPIC9 vector; lane 5, culture supernatant from cells harboring pPIC9-OfHex1 vector. (B) Gel filtration chromatography of the recombinant OfHex1 on Superdex 200 10/30 GL column.
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Table 1 Purification efficiency of the recombinant OfHex1. Purification steps
Total protein (mg)
Total activity (U)a
Specific activity (U/mg)
Overall yield (%)
Overall purification (fold)
Culture media Ammonium sulfate IMAC Sepharose HP Q Sepharose HP
175.5 67.7 9.77 7.67
362.16 301.68 222.75 182.88
2.06 4.46 22.79 23.85
100 83.3 61.5 50.5
1 2.16 11.04 11.56
a
One unit represents the amount of 1 lmol pNP released per minute.
Characterization of the recombinant OfHex1 The highest activity of the recombinant OfHex1 was observed at pH 7.0 (Fig. 3A) and at 30 °C (Fig. 3B). These properties were consistent with that of the native OfHex1 [6]. The substrate specificity of the recombinant OfHex1 was first examined by using four 4MU-derivated substrates (Fig. 4A). The recombinant OfHex1 could release 4MU from both 4MU-b-GlcNAc and 4MU-b-GalNAc. It showed a preference for the substrate with gluco-configuration, which was similar to the native OfHex1. The recombinant OfHex1 also could not hydrolyze 4MU-b-Glc at all. Charged substrate 4MU-b-GlcNAc-6-SO4 was applied. The hydrolytic activity of the recombinant OfHex1 toward 4MU-b-GlcNAc-6SO4 was 156 times lower than toward 4MU-b-GlcNAc. The substrate specificity of the recombinant OfHex1 was also examined by substrates (GlcNAc)2 and GnGn-PA. After 5 min incu-
Fig. 4. Substrate specificity evaluation of the recombinant OfHex1. (A) 4MUderivated substrates. The liberation of 4MU was measured at the excitation wavelength of 360 nm and the emission wavelength of 450 nm. (B) (GlcNAc)2. The hydrolysis product was detected by HPLC analysis by a TSKgel Amide-80 column.
bation of (GlcNAc)2 with the recombinant OfHex1, enzymatic hydrolysis product GlcNAc could be easily detected by HPLC (Fig. 4B). But even 1 h incubation of GnGn-PA with the same amount of the recombinant OfHex1, no enzymatic hydrolysis product could be detected (data not shown).
Discussion
Fig. 3. Effects of pH and temperature on the recombinant OfHex1’s activity. (A) Optimum pH. (B) Optimum temperature. The enzyme activity was determined by using the substrate, pNP-b-GlcNAc, and measuring the liberation of pNP at 405 nm.
b-N-acetyl-D-hexosaminidases are involved in many important physiological processes in insect ranging from chitin degradation, N-glycan modification, glycoconjugates degradation and egg– sperm interaction [16]. The specific inhibitors against insect b-Nacetyl-D-hexosaminidases show potency as drugs to block specific physiological functions. Large amounts of insect b-N-acetyl-Dhexosaminidases are therefore required for structural and mechanism studies and drug development. OfHex1 is the insect b-N-acetyl-D-hexosaminidase whose physiological function is presumed to be only involved in chitin degradation [6]. This property makes OfHex1 be a potential target for pesticide design. To evaluate whether the recombinant OfHex1 can fully reproduce the property of the native OfHex1, we characterized the recombinant OfHex1 in detail. The recombinant OfHex1 has a similar specific activity, the same optimal pH and optimal temperature as the native OfHex1 [6]. It demonstrates the C-terminal 6His tag does not affect activity. The substrate specificity of the recombinant OfHex1 suggested that it can release 4MU from both 4MU-b-GlcNAc and 4MU-b-GalNAc with a slightly preference for the substrate with gluco-configuration. It can efficiently degrade the chitooligosaccharide substrate (GlcNAc)2 but can not
T. Liu et al. / Protein Expression and Purification 68 (2009) 99–103
hydrolyze the N-glycan substrate GnGn-PA. The low activity of the recombinant OfHex1 toward substrate 4MU-b-GlcNAc-6-SO4 suggests that the negative charge at 6-OH on the substrate has obvious side effect on the enzyme activity. In conclusion, we have successfully expressed, purified and characterized the recombinant insect b-N-acetyl-D-hexosaminidase, OfHex1, which shows promising potential as a specific target for the development of pesticides. The specific properties of the native OfHex1 are well reproduced in the recombinant OfHex1. This work provides a low-costing alternative for the preparation of an insect b-N-acetyl-D-hexosaminidase for biochemical studies, drug development as well as biotechnological application. Acknowledgments Financial supports were provided by National Natural Science Foundation of China (20536010), the State Key Laboratory of Bio-Organic & Natural Products Chemistry (Shanghai, China), the National Special Fund for State Key Laboratory of Bioreactor Engineering (ECUST) (Shanghai, China), the Key Laboratory of Natural Pesticide & Chemical Biology of Ministry of Education (Wuhan, China) and Shanghai Key Lab of Chemical Biology (China). References [1] H. Merzendorfer, L. Zimoch, Chitin metabolism in insects: structure, function and regulation of chitin synthases and chitinases, J. Exp. Biol. 206 (2003) 4393– 4412. [2] T. Fukamizo, K.J. Kramer, Mechanism of chitin hydrolysis by the binary chitinase system in insect moulting fluid, Insect Biochem. 15 (1985) 141–145. [3] E. Cohen, Chitin synthesis and degradation as targets for pesticide action, Arch. Insect Biochem. Physiol. 22 (1993) 245–261.
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[4] K.J. Kramer, S. Muthukrishnan, Insect chitinases: molecular biology and potential use as biopesticides, Insect Biochem. Mol. Biol. 27 (1998) 887– 900. [5] N. Dahiya, R. Tewari, G.S. Hoondal, Biotechnological aspects of chitinolytic enzymes: a review, Appl. Microbiol. Biotechnol. 71 (2006) 773–782. [6] Q. Yang, T. Liu, F. Liu, M. Qu, X. Qian, A novel beta-N-acetyl-D-hexosaminidase from the insect Ostrinia furnacalis (Guenée), FEBS J. 275 (2008) 5690–5702. [7] F. Altmann, H. Schwihla, E. Staudacher, J. Glössl, L. März, Insect cells contain an unusual, membrane-bound beta-N-acetylglucosaminidase probably involved in the processing of protein N-glycans, J. Biol. Chem. 270 (1995) 17344–17349. [8] R. Léonard, D. Rendic, C. Rabouille, I.B. Wilson, T. Préat, F. Altmann, The Drosophila fused lobes gene encodes an N-acetylglucosaminidase involved in Nglycan processing, J. Biol. Chem. 281 (2006) 4867–4875. [9] C. Geisler, J.J. Aumiller, D.L. Jarvis, A fused lobes gene encodes the processing beta-N-acetylglucosaminidase in Sf9 cells, J. Biol. Chem. 283 (2008) 11330– 11339. [10] J.J. Aumiller, J.R. Hollister, D.L. Jarvis, Molecular cloning and functional characterization of beta-N-acetylglucosaminidase genes from Sf9 cells, Protein Expr. Purif. 47 (2006) 571–590. [11] T. Okada, S. Ishiyama, H. Sezutsu, A. Usami, T. Tamura, K. Mita, K. Fujiyama, T. Seki, Molecular cloning and expression of two novel beta-Nacetylglucosaminidases from silkworm Bombyx mori, Biosci. Biotechnol. Biochem. 71 (2007) 1626–1635. [12] N. Tomiya, S. Narang, J. Park, B. Abdul-Rahman, O. Choi, S. Singh, J. Hiratake, K. Sakata, M.J. Betenbaugh, K.B. Palter, Y.C. Lee, Purification, characterization, and cloning of a Spodoptera frugiperda Sf9 beta-N-acetylhexosaminidase that hydrolyzes terminal N-acetylglucosamine on the N-glycan core, J. Biol. Chem. 281 (2006) 19545–19560. [13] F. Cattaneo, M. Ogiso, M. Hoshi, M.E. Perotti, M.E. Pasini, Purification and characterization of the plasma membrane glycosidases of Drosophila melanogaster spermatozoa, Insect Biochem. Mol. Biol. 32 (2002) 929–941. [14] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding, Anal. Biochem. 72 (1976) 248–254. [15] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680–685. [16] J. Intra, G. Pavesi, D.S. Horner, Phylogenetic analyses suggest multiple changes of substrate specificity within the glycosyl hydrolase 20 family, BMC Evol. Biol. 8 (2008) 214.
Contents lists available at ScienceDirect
Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep
Expression, purification and characterization of the chitinolytic b-N-acetyl-D-hexosaminidase from the insect Ostrinia furnacalis Tian Liu a,1, Fengyi Liu a,1, Qing Yang a,b,*, Jun Yang a a b
Department of Bioscience and Biotechnology, Dalian University of Technology, Dalian 116024, China State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China
a r t i c l e
i n f o
Article history: Received 8 May 2009 and in revised form 3 June 2009 Available online 9 June 2009 Keywords: b-N-acetyl-D-hexosaminidase Chitin degradation Expression Insect
a b s t r a c t Insect b-N-acetyl-D-hexosaminidases are of particular interest due to their multiple physiological roles in many life processes. Chitinolytic b-N-acetyl-D-hexosaminidases, which function only in chitin degradation in insects, have long been regarded as species-specific target potentials in developing environmental friendly pesticides. Here the chitinolytic b-N-acetyl-D-hexosaminidase from the insect Ostrinia furnacalis was cloned and expressed in the yeast strain, Pichia pastoris, to meet the demands of biochemical studies and drug development. Enzymatic assay as well as Western blot confirmed that the high-level expression could be achieved after the induction of methanol for 120 h. Through the sequential combination of ammonium sulfate precipitation, metal chelating chromatography as well as anion exchange chromatography, 7.7 mg of the recombinant OfHex1 with high purity was obtained from 1 liter of culture supernatant. The recombinant OfHex1, characterized as a homodimer with molecular weight of 130 kDa, exhibited the same enzymatic activities as its native form, which could efficiently degrade the chitooligosaccharide substrate (GlcNAc)2 and release 4-methylumbelliferone (4MU) from substrates, 4MU-b-GlcNAc and 4MU-b-GalNAc. This work provides a low-costing and high-efficient purification procedure for the preparation of insect b-N-acetyl-D-hexosaminidases. Ó 2009 Elsevier Inc. All rights reserved.
Introduction Chitin biodegradation exists in chitin-containing organisms such as fungi, crustaceans and insects, whose main structural component is chitin, and in some bacteria which utilize chitin as carbon and nitrogen sources. Two enzymes play key roles in this process: chitinase (EC 3.2.1.14) firstly catalyzes the degradation of chitin into chitooligosaccharides, and b-N-acetyl-D-hexosaminidase (EC 3.2.1.52) hydrolyzes chitooligosaccharides into N-acetyl2 D-glucosamine (GlcNAc) [1]. The ratio of these two enzymes regulates the efficiency of chitin degradation and thus affects remodeling of chitin components of insects and fungi [2]. In this regard, these enzymes could be specific target potentials in the development of environmental friendly pesticides [3,4]. On the other hand, since chitin is the second most abundant but underutilized biomass in nature, the degradation of chitin by chitinolytic enzymes is of biotechnological importance [5].
* Corresponding author. Address: Department of Bioscience and Biotechnology, Dalian University of Technology, No. 2 Linggong Road, Dalian 116024, China. Fax: +86 0411 84707245. E-mail address: [email protected] (Q. Yang). 1 These authors contributed equally to this work. 2 Abbreviations used: 4MU, 4-methylumbelliferone; GlcNAc, N-acetyl-D-glucosamine; GST, glutathione-S-transferase; TBS, tris buffer saline. 1046-5928/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2009.06.004
We previously reported an insect b-N-acetyl-D-hexosaminidase from the Asian corn borer, Ostrinia furnacalis (Guenée), designated as OfHex1 [6]. Unlike other b-N-acetyl-D-hexosaminidases which function in N-glycan modification [7–9], glycoconjugate degradation [10,11] or sperm–egg recognition [12], OfHex1 was identified as the first insect b-N-acetyl-D-hexosaminidase whose physiological role was only involved in chitin degradation. Therefore, the preparation of this enzyme is of significance in the view of biochemical studies and biotechnology. Three reports associated with the recombinant expression and purification of insect b-N-acetyl-D-hexosaminidases have been revealed using insect cell lines as expression system, in which the b-N-acetyl-D-hexosaminidase activity of the host cell could not be ruled out. The glutathione-S-transferase (GST) tagged Sf-FDL from Spodoptera frugiperda and Dm-FDL from Drosophila melanogaster were expressed in Sf9 cells and purified by GST affinity chromatography [9]. It is the only example of successful expression and purification of recombinant insect b-N-acetyl-D-hexosaminidases. The SfHex, SfGlcNAcase-1 and SfGlcNAcase-3 from S. frugiperda were expressed in Sf9 cells but failed to be purified [10,13]. Since yeast is the low-cost expression system for producing large quantities of eukaryotic proteins, the recombinant expression of insect b-N-acetyl-D-hexosaminidases in yeast was tried. FDL, HEXO1 and HEXO2 from D. melanogaster were cloned into the expression vector, pPICZaA, and expressed in Pichia pastoris GS115 without purification
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[8]. BmGlcNAcase1 and BmGlcNAcase2 from Bombyx mori were cloned into the expression vectors, pPICZaB and pPICZaC, respectively, and expressed in P. pastoris GS115, but purification attempts were still unsuccessful [8,11]. Herein we report the insect b-N-acetyl-D-hexosaminidase, OfHex1, was cloned into the expression vector pPIC9 instead of pPICZa series and expressed in the yeast strain, P. pastoris GS115. Through two chromatographic separation steps, the recombinant OfHex1 with high purity was obtained. To our knowledge, this is the first example of successful expression and purification of insect b-N-acetyl-D-hexosaminidase in yeast system. Partial enzymatic characterization was also performed.
Materials and methods Materials Restrict enzymes were purchased from NEB (Ipswich, MA). p-Nitrophenyl-N-acetyl-b-D-glucosaminide, 4-methylumbelliferyl4-methylumbelliferyl-N-acetyl-b-D-glucosb-D-glucopyranoside, aminide, 4-methylumbelliferyl-N-acetyl-b-D-galactosaminide and (GlcNAc)2 were purchased from Sigma–Aldrich (St. Louis, MO). 4Methylumbelliferyl-6-sulfo-N-acetyl-b-D-glucosaminide, HisTagTM monoclonal antibody and rabbit anti mouse peroxidase-conjugated secondary antibody were purchased from Merck (San Diego, CA). (GlcNAcb1,2Mana1,6)(GlcNAcb1,2Mana1,3)Manb1,4GlcNAcb1,4 GlcNAc-PA was purchased from Takara, (Dalian, China). All the other chemicals were purchased from commercial sources with highest purity.
Enzyme activity and protein concentration assay The enzyme activity was measured using p-nitrophenyl-N-acetyl-b-D-glucosaminide (pNP-b-GlcNAc) as substrate [6]. Briefly, the reaction was stopped by adding an equal volume of 0.5 M Na2CO3 and absorbance at 405 nm was monitored by microplate reader (TECAN, Männedorf, Switzerland). The protein concentration was assayed by the method of Bradford [14]. Purification of the recombinant OfHex1 All the column chromatographies were performed by using ÄKTA Purifier system (GE Healthcare, Uppsala, Sweden). In the first step, solid ammonium sulfate was added to OfHex1-expressed supernatant to 65% saturation. After incubation at 4 °C for 1 h, the medium supernatant was centrifuged at 8000g for 30 min. Then the pellet was dissolved in buffer A (20 mM sodium phosphate (pH 7.4), containing 0.1 M NaCl) and centrifuged at 12,000g for 10 min. The supernatant was loaded onto an IMAC Sepharose High Performance column (20 ml, GE Healthcare, Uppsala, Sweden) pre-equilibrated with buffer A. The column was first washed with buffer A containing 75 mM imidazole and the 6His tagged OfHex1 was eluted with buffer A containing 150 mM imidazole. Then the sample was desalted by a HiTrap desalting column (53 ml, GE Healthcare, Uppsala, Sweden) with buffer B (20 mM bis–Tris, pH 6.5) and loaded onto a Q Sepharose High Performance column (10 ml, GE Healthcare, Uppsala, Sweden) pre-equilibrated with buffer B. After washing, the column was eluted with a linear gradient of NaCl from 75 to 275 mM in buffer B. Fractions (4 ml each) were collected and enzymatic activities were assayed. The purity was analyzed by SDS–PAGE.
Construction of expression plasmids SDS–PAGE and gel filtration chromatography The vector CTB557-2-1 carrying the gene encoding OfHex1 was stored in our lab. Based on the cDNA sequence of OfHex1 (GenBank Accession No. DQ887769), two primers, 50 -GCTTACGTAGAATTC GAGGACGTAGTATGGCGCTGGT-30 and 50 -TTAATTCGCGGCCGC TTA ATGATGATGATGATGATGCGAGTAACAGTACCCCTC-30 were synthesized to amplify gene encoding the mature OfHex1 by PCR from CTB557-2-1. Underlined bases correspond to restriction enzymes sequences of EcoRI/NotI, respectively, and bases in bold correspond to the sequence encoding 6His tag at C-terminal. The resulting PCR products were digested with EcoRI/NotI and then cloned into EcoRI/NotI cut pPIC9 vector (Invitrogen, Carlsbad, CA) to generate the expression plasmid named pPIC9-OfHex1.
SDS–PAGE was carried out as described by Laemmli [15], using 10% (w/v) polyacrylamide resolving gel and 4% (w/v) polyacrylamide stacking gel. After electrophoresis, proteins in gel were visualized with Coomassie Brilliant Blue. For analytical gel filtration, 50 ll of purified OfHex1 was applied to Superdex 200 10/30 GL column (GE Healthcare, Uppsala, USA) pre-equilibrated with 20 mM of sodium phosphate (pH 6.8) containing 150 mM NaCl. The column was eluted at a flow rate of 0.3 ml/min. The molecular weight of the recombinant OfHex1 was determined as described before [6]. Western blot
Transformation and expression of the recombinant OfHex1 The expression vector plasmid (pPIC9-OfHex1) was linearized using PmeI and electroporated into the P. pastoris GS115 cells. The selection of His+ and Mut+ P. pastoris transformant was performed according to the manufacturer’s instructions. Positive transformants were cultured in 2 ml of BMGY broth (2% peptone, 1% yeast extract, 1% glycerol, 1.34% yeast nitrogen base and 0.1 M potassium phosphate (pH 6.0)) in tubes for 24 h and then induced for 48 h by the addition of 1% of methanol at every 12 h intervals. The transformant with the highest enzyme activities in the culture supernatant was selected. The selected transformant was precultured in 100 ml of BMGY broth and then transferred to 1 L of BMMY broth (2% peptone, 1% yeast extract, 1% methanol, 1.34% yeast nitrogen base and 0.1 M potassium phosphate (pH 6.0)). The cultures were incubated at 29 °C with continuous shaking and methanol (1% of the total volume) was re-added every 24 h. After 120 h fermentation, the supernatant was collected by centrifugation at 8000g for 30 min.
The proteins were separated by 10% SDS–PAGE and transferred onto a polyvinylidene fluoride membrane. The membrane was blocked with 5% milk in Tris buffer saline (TBS) for 1 h and then incubated with HisTagTM monoclonal antibody (Merck, San Diego, CA) at a dilution of 1:1000 in 3% milk in TBS overnight at 4 °C. After washing, the membrane was incubated with a rabbit anti mouse peroxidase-conjugated secondary antibody (Merck, San Diego, CA) at a dilution of 1:7500 in 3% milk in TBS for 1 h. The blot was developed with 3,3-diaminobenzidine tetrahydrochloride solution. Characterization of the recombinant OfHex1 The determination of the optimal pH was performed in 100 mM Britton-Robinson’s wide range buffer from pH 3 to pH 11 with one pH scale interval using pNP-b-GlcNAc as substrate. For the determination of the optimal reaction temperature, the reaction mixtures were incubated at various temperatures from 10 °C to 60 °C with 10 °C interval.
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To characterize the substrate spectrum of the recombinant OfHex1, enzymatic activities were measured using different kinds of substrates. First, the 4-methylumbelliferone (4MU)-linked substrates including 4-methylumbelliferyl-b-D-glucopyranoside (4MU-b-Glc), 4-methylumbelliferyl-N-acetyl-b-D-glucosaminide (4MU-b-GlcNAc), 4-methylumbelliferyl-N-acetyl-b-D-galactosaminide (4MU-b-GalNAc) and 4-methylumbelliferyl-6-sulfo-N-acetylb-D-glucosaminide (4MU-b-GlcNAc-6-SO4) were applied. The reaction sample consisted of 175 ll of 20 mM bis–Tris buffer (pH 6.5), 20 ll of 1 mM 4MU-linked substrates and 5 ll of enzyme. After incubation at 25 °C for 5 min, 1 ml of 0.5 M Na2CO3 was added to the reaction sample. The released 4MU was monitored by F-4500 spectrofluorimeter (HITACHI, Tokyo, Japan) with excitation wavelength of 360 nm and emission wavelength of 450 nm. Second, the chitooligosaccharide substrate (GlcNAc)2 and the pyridylaminated N-glycan substrate (GlcNAcb1,2Mana1,6)(GlcNAcb1,2Mana1,3)Manb1,4GlcNAcb1,4GlcNAc-PA (GnGn-PA) were applied. The hydrolytic efficiencies of (GlcNAc)2 and GnGn-PA were measured by HPLC using TSKgel Amide-80 column (4.6 250 mm, Tosoh, Tokyo, Japan) and a Hypersil ODS2 column (4.6 250 mm, Thermo, Waltham, USA), respectively [6].
Results Cloning and expression of the recombinant OfHex1 The gene encoding mature OfHex1 was inserted into the yeast expression vector pPIC9. The a-factor signal sequence carried by the vector was used to promote secretory expression. A 6His tag was added to the C-terminal to facilitate purification. Correct insertion and reading frame of OfHex1 were confirmed by DNA sequencing. Then the resulting pPIC9-OfHex1 vector was linearized and electroporated into P. pastoris GS115 cells. The positive transformants were obtained by screening His+/Mut+ plates. The His+/Mut+ P. pastoris transformant was cultured and OfHex1 expression was induced by adding 1% methanol. OfHex1 expression was monitored every 12 h by measuring both b-N-acetyl-Dhexosaminidase activity and protein concentration in culture supernatant. The specific activity in culture supernatant was determined and used to determine the optimal induction time. As shown in Fig. 1, the highest specific activity with 2.06 U/mg of pro-
Fig. 1. Total activities and specific activities in the culture supernatant at different time of induction. The black squares and white squares represent total activities and specific activities in 1 L of culture supernatant. The enzyme activity was determined by using the substrate, pNP-b-GlcNAc, and measuring the liberation of pNP at 405 nm.
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tein was observed after the induction for 120 h, when 362 U of total activity were obtained in 1 L of culture supernatant. Purification of the recombinant OfHex1 The recombinant OfHex1 was purified to homogeneity from the culture supernatant by a sequential combination of ammonium sulfate precipitation, metal chelating as well as anion exchange chromatographies (Fig. 2A). Through ammonium sulfate precipitation, the sample was not only concentrated from 1 L to 20 ml but also purified by 2.16 folds (Fig. 2A, lane 1). The first-step column chromatography, Ni2+-chelating affinity chromatography, contributed to 5-fold purification efficiency (Table 1). By washing the column with buffer containing 75 mM of imidazole before eluting the target protein, most of the contaminating proteins were removed (Fig. 2A, lane 2). Then the residual impurities were removed by anion exchange chromatography (Fig. 2A, lane 3). About 7.7 mg of purified recombinant OfHex1 was obtained from 1 L of culture supernatant. The total purification efficiency was 11 folds, which was summarized in Table 1. The specific activity of the purified recombinant OfHex1 (23.85 U/mg) was similar to specific activity of the native OfHex1 (24.96 U/mg) [6]. The purified protein was resolved as a single band with molecular weight of 69 kDa by SDS–PAGE and verified by Western blot using anti-His tag antibody (Fig. 2A, lanes 3 and 5). The recombinant OfHex1 was also resolved as a single sharp peak by analytical gel filtration chromatography (Fig. 2B), whose molecular weight was determined to be 130 kDa by standard curve. Thus, the recombinant OfHex1 was in the same form of homodimer as the native OfHex1 [6].
Fig. 2. Purification of the recombinant OfHex1. (A) Lanes 1–3, SDS–PAGE analysis of purification efficiency of the recombinant OfHex1. Lane 1, sample after ammonium sulfate precipitation; lane 2, sample after metal chelate chromatography on IMAC Sepharose HP column; lane 3, the purified recombinant OfHex1 after anion exchange chromatography on Q Sepharose HP column. Lanes 4 and 5, Western blot (WB) of the recombinant OfHex1 by using HisTagTM monoclonal antibody. Lane 4, culture supernatant from cells harboring pPIC9 vector; lane 5, culture supernatant from cells harboring pPIC9-OfHex1 vector. (B) Gel filtration chromatography of the recombinant OfHex1 on Superdex 200 10/30 GL column.
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Table 1 Purification efficiency of the recombinant OfHex1. Purification steps
Total protein (mg)
Total activity (U)a
Specific activity (U/mg)
Overall yield (%)
Overall purification (fold)
Culture media Ammonium sulfate IMAC Sepharose HP Q Sepharose HP
175.5 67.7 9.77 7.67
362.16 301.68 222.75 182.88
2.06 4.46 22.79 23.85
100 83.3 61.5 50.5
1 2.16 11.04 11.56
a
One unit represents the amount of 1 lmol pNP released per minute.
Characterization of the recombinant OfHex1 The highest activity of the recombinant OfHex1 was observed at pH 7.0 (Fig. 3A) and at 30 °C (Fig. 3B). These properties were consistent with that of the native OfHex1 [6]. The substrate specificity of the recombinant OfHex1 was first examined by using four 4MU-derivated substrates (Fig. 4A). The recombinant OfHex1 could release 4MU from both 4MU-b-GlcNAc and 4MU-b-GalNAc. It showed a preference for the substrate with gluco-configuration, which was similar to the native OfHex1. The recombinant OfHex1 also could not hydrolyze 4MU-b-Glc at all. Charged substrate 4MU-b-GlcNAc-6-SO4 was applied. The hydrolytic activity of the recombinant OfHex1 toward 4MU-b-GlcNAc-6SO4 was 156 times lower than toward 4MU-b-GlcNAc. The substrate specificity of the recombinant OfHex1 was also examined by substrates (GlcNAc)2 and GnGn-PA. After 5 min incu-
Fig. 4. Substrate specificity evaluation of the recombinant OfHex1. (A) 4MUderivated substrates. The liberation of 4MU was measured at the excitation wavelength of 360 nm and the emission wavelength of 450 nm. (B) (GlcNAc)2. The hydrolysis product was detected by HPLC analysis by a TSKgel Amide-80 column.
bation of (GlcNAc)2 with the recombinant OfHex1, enzymatic hydrolysis product GlcNAc could be easily detected by HPLC (Fig. 4B). But even 1 h incubation of GnGn-PA with the same amount of the recombinant OfHex1, no enzymatic hydrolysis product could be detected (data not shown).
Discussion
Fig. 3. Effects of pH and temperature on the recombinant OfHex1’s activity. (A) Optimum pH. (B) Optimum temperature. The enzyme activity was determined by using the substrate, pNP-b-GlcNAc, and measuring the liberation of pNP at 405 nm.
b-N-acetyl-D-hexosaminidases are involved in many important physiological processes in insect ranging from chitin degradation, N-glycan modification, glycoconjugates degradation and egg– sperm interaction [16]. The specific inhibitors against insect b-Nacetyl-D-hexosaminidases show potency as drugs to block specific physiological functions. Large amounts of insect b-N-acetyl-Dhexosaminidases are therefore required for structural and mechanism studies and drug development. OfHex1 is the insect b-N-acetyl-D-hexosaminidase whose physiological function is presumed to be only involved in chitin degradation [6]. This property makes OfHex1 be a potential target for pesticide design. To evaluate whether the recombinant OfHex1 can fully reproduce the property of the native OfHex1, we characterized the recombinant OfHex1 in detail. The recombinant OfHex1 has a similar specific activity, the same optimal pH and optimal temperature as the native OfHex1 [6]. It demonstrates the C-terminal 6His tag does not affect activity. The substrate specificity of the recombinant OfHex1 suggested that it can release 4MU from both 4MU-b-GlcNAc and 4MU-b-GalNAc with a slightly preference for the substrate with gluco-configuration. It can efficiently degrade the chitooligosaccharide substrate (GlcNAc)2 but can not
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hydrolyze the N-glycan substrate GnGn-PA. The low activity of the recombinant OfHex1 toward substrate 4MU-b-GlcNAc-6-SO4 suggests that the negative charge at 6-OH on the substrate has obvious side effect on the enzyme activity. In conclusion, we have successfully expressed, purified and characterized the recombinant insect b-N-acetyl-D-hexosaminidase, OfHex1, which shows promising potential as a specific target for the development of pesticides. The specific properties of the native OfHex1 are well reproduced in the recombinant OfHex1. This work provides a low-costing alternative for the preparation of an insect b-N-acetyl-D-hexosaminidase for biochemical studies, drug development as well as biotechnological application. Acknowledgments Financial supports were provided by National Natural Science Foundation of China (20536010), the State Key Laboratory of Bio-Organic & Natural Products Chemistry (Shanghai, China), the National Special Fund for State Key Laboratory of Bioreactor Engineering (ECUST) (Shanghai, China), the Key Laboratory of Natural Pesticide & Chemical Biology of Ministry of Education (Wuhan, China) and Shanghai Key Lab of Chemical Biology (China). References [1] H. Merzendorfer, L. Zimoch, Chitin metabolism in insects: structure, function and regulation of chitin synthases and chitinases, J. Exp. Biol. 206 (2003) 4393– 4412. [2] T. Fukamizo, K.J. Kramer, Mechanism of chitin hydrolysis by the binary chitinase system in insect moulting fluid, Insect Biochem. 15 (1985) 141–145. [3] E. Cohen, Chitin synthesis and degradation as targets for pesticide action, Arch. Insect Biochem. Physiol. 22 (1993) 245–261.
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