Protein Expression and Purification 60 (2008) 221–224
Contents lists available at ScienceDirect
Protein Expression and Purification j o u r n a l h o m e p a g e : w w w . e l s e v i e r. c o m / l o c a t e / y p r e p
Expression, purification and characterization of pectin methylesterase inhibitor from kiwi fruit in Escherichia coli Yanling Hao 1, Xinyi Huang 1, Xiaohong Mei, Ruoyu Li, Zhengyuan Zhai, Sheng Yin, Ying Huang, Yunbo Luo * College of Food Science and Nutritional Engineering, China Agricultural University, 17 Qing Hua East Road, Hai Dian District, Beijing 100083, PR China
a r t i c l e
i n f o
Article history: Received 4 March 2008 Received in revised form 14 April 2008 Available online 26 April 2008 Keywords: Pectin methylesterase inhibitor Escherichia coli Expression Purification Activity analysis
a b s t r a c t A significant problem in production of fruit juices for human consumption is auto-clarification, where enzyme catalyzes pectin demethylation resulting in loss of the ‘‘natural” cloudy appearance of juices. To overcome this problem, a plant inhibitor protein which blocks the action of pectin methylesterase has been used. In this paper, expression of recombinant kiwi pectin methylesterase inhibitor (PMEI) was carried out in Escherichia coli, and the target protein was expressed in the form of inclusion bodies. The expression level reached 46% of total cell protein. Then the fusion protein was purified by nickel ion metal affinity chromatography, and the purity was finally up to 98%. After refolding in GSH/GSSG redox system, recombinant PMEI not only could efficiently inhibit PMEs from eight different plants, but could remain effective inhibitor activity in the pH 3.0–10.0 and 20–40 °C. Thus, recombinant PMEI has potential applica tion in the production of fruit juices product industry. © 2008 Published by Elsevier Inc.
One of the main problems in the fruit product industry is the maintenance of the turbidity in fruit and vegetable juices during the process and storage. Pectin demethylation by endogenous pec tin methylesterase (PME; EC188.8.131.52)2 is considered as the main cause of cloud loss . Now, in order to overcome cloud loss, the thermal treatment is widely used to inactivate PME in juice manu facturing. But the thermal process will severely deteriorate nutri tional ingredients and sensory attributes of juices. In 1990, Balestrieri et al. found a pectin methylesterase inhibitor (PMEI) in ripe kiwi fruit , which comprises 152 amino acid resi dues, accounting for a molecular weight of 16.277 kDa . Through forming 1:1 non-covalent complex to cover active site, the kiwi PMEI can completely inhibit plant PMEs activity . Compared with thermal inactivation of PMEs, such milder treatment could remain better flavor quality and higher vitamin content . Casst aldo et al. successfully utilized the kiwi PMEI to prolong orange juice shelf life to 9 months at 5 °C . In addition, affinity chroma tography on resin-bound PMEI can be used to concentrate and detect residual PME activity in fruit and vegetable products [7,8]. The discovery of kiwi PMEI provided a new way to solve fruit juice cloud loss. But, so far, the PMEI can only be obtained from kiwi fruit. Furthermore, the extracting yield from kiwi fruit is
rather low. This greatly limited the application of PMEI in the juice industry. In order to conveniently obtain PMEI, gene engineering strain was taken into account. To date, E. coli has been widely used as the host strain to produce heterologous proteins due to its rapid growth rate, continuous fermentation capacity and relatively low cost . Consequently, in this work, we attempted to produce recombinant PMEI on a large scale using E. coli expression system. Materials and methods Materials Escherichia coli DH5a was obtained from TianGen (Beijing, China). E. coli BL21 (DE3) and vector pET-30a (+) that produces a fusion protein with His-Tag, was purchased from Novagen (Mad ison, USA). Plasmid pPIC9K/kWPMEI containing kiwi fruit PMEI gene was constructed as described previously . T4 DNA ligase and restriction endonucleases were purchased from TaKaRa (Dalian, China). Citrus pectin (70% methylesterified), isopropylb-d-thiogalactopyranoside (IPTG) and ruthenium red dye were obtained from Sigma (St. Louis, MO). Construction of fusion expression vector
* Corresponding author. Fax: +86 10 62736479. E-mail address: [email protected]
(Y. Luo). 1 These authors contributed equally. 2 Abbreviations used: PME, pectin methylesterase; PMEI, pectin methylesterase inhibitor; E. coli, Escherichia coli; IPTG, isopropyl-b-d-thiogalactopyranoside; GSH, l-Glutathione (reduced); GSSG, l-Glutathione (oxidized); SDS–PAGE, sodium dode cyl sulfate–polyacrylamide gel electrophoresis; LB, Luria–Bertani. 1046-5928/$ – see front matter © 2008 Published by Elsevier Inc. doi:10.1016/j.pep.2008.04.004
The cDNA encoding kiwi fruit PMEI in the recombinant plasmid pPIC9K/kWPMEI was cloned into the fusion expression vector pET30a (+) as a EcoRI–NotI fragment. The recombinant plasmid, desig nated as pET30a-PMEI, was screened in DH5a. Then pET30a-PMEI was transformed into the E. coli strain BL21 (DE3) by heat shock.
Y. Hao et al. / Protein Expression and Purification 60 (2008) 221–224
Expression of the PMEI in E. coli
A single-colony transformant was inoculated into LB medium containing 50 lg/mL kanamycin and grown at 37 °C. The over night culture was 50-fold diluted to inoculate in 100 mL fresh LB medium. When the optical density (OD600) reached about 0.6, IPTG was then added to a final concentration of 1 mM to induce expression of fusion protein for 4 h at 37 °C . Cell pellets har vested were suspended in 1 mL PBS buffer (pH 7.4) and were sub jected to sonication in ice bath at 300 W with 10 s on/off cycle to prevent overheating, for a total time of 6 min. The total cellular proteins were then partitioned into soluble and insoluble frac tions by centrifugation at 12,000g for 10 min at 4 °C. In order to determine the solubility of fusion protein, the samples were ana lyzed by SDS–PAGE.
Construction of the fusion expression vector
Purification and refolding of recombinant PMEI The inclusion bodies in insoluble fraction were washed twice with buffer containing 50 mM Tris–HCl (pH 8.0), 100 mM NaCl and 0.5% (v/v) Triton X-100. The washed inclusion bodies were fur ther dissolved in binding buffer containing 20 mM sodium phos phate, 0.5 M NaCl, 20 mM imidazole, 20 mM b-ME and 8.0 M urea (pH 7.4). After solubilization, the recombinant protein was puri fied by affinity chromatography. Ni sepharose 6 Fast Flow media was packed and equilibrated with binding buffer. The target pro tein was eluted with a stepwise gradient of imidazole from 20 to 200 mM. Purified proteins were refolded by urea gradient dialysis at 4 °C in PBS buffer containing 1 mM EDTA, 1% glycine, 5% glyc erol, 0.9 mM GSH, 0.1 mM GSSG. Meanwhile, urea was added into the PBS buffer every 12 h at a gradient concentration (6, 4, 2, 1, 0.5 and 0 M).
The whole encoding region of kiwi fruit PMEI was cloned into the fusion expression vector pET-30a to generate the recombinant plasmid pET30a-PMEI. The deduced amino acid sequence of recombinant PMEI showed 98% homology with the native PMEI which was purified from kiwi fruit. There were two different amino acids (63 Thr ! Ala and 123 Asn ! Asp) between the native PMEI and recombinant PMEI. Expression and purification of PMEI The expression of fusion protein was induced with 1 mM IPTG at 37 °C for 4 h. After sonication and centrifugation of bac teria cells, the soluble and insoluble fractions were analyzed by SDS–PAGE. The results showed that recombinant protein was only found in the insoluble fraction (Fig. 1), which indicated that the recombinant proteins were expressed in the form of inclu sion bodies. SDS–PAGE analysis revealed that the target protein accumulated up to 46% of the total cell protein. After nickel ion metal affinity chromatography, the purity of the recombinant proteins reached 98% (Fig. 1). In order to provide evidence for correct refolding of the recombination protein, in vitro activity of the PMEI were tested. In the experiment, purified proteins refolded in PBS buffer containing only 1 mM EDTA, 1% glycine and 5% glycerol showed no biological activity. However, when 0.9 mM GSH and 0.1 mM GSSG were added into the refolding buffer, the recombinant PMEI could efficiently inhibit PMEs from eight different plants. So, the presentation of the reducing and oxidizing agents (GSH/GSSH) is essential for PMEI inclusion body refolding.
Biological activity assay of recombinant PMEI The biological activity of recombinant PMEI was measured in vitro according to its inhibitory effect on PMEs from eight different plants (tomato, orange, carrot, apple, pineapple, hawthorn, grape and kiwi). Plant fruit PME extraction was prepared according to Giovane et al. . Inhibitory capacity of purified recombinant PMEI to PMEs from different plants was measured using a gel diffu sion assay . Gel containing 0.1% pectin substrate was prepared at pH 7.0. Wells were punched in the gel. Excess amount of purified recombinant PMEI was, respectively, mixed with a certain amount of PME crude extractions at room temperature for 15 min. Then, the mixtures were placed into each well, and the whole well was sealed and incubated at 30 °C for 16 h. After incubation, the gel was stained with 0.05% (w/v) ruthenium red dye for 45 min. Mean while, the same amount of the PMEs from eight fruits was respec tively assayed using the same method without any recombinant PMEI.
Inhibitory effect of purified recombinant PMEI on PME from different plants The inhibitory capacity of recombinant PMEI to PMEs was mea sured using a gel diffusion assay. The results showed that stained zones were around the wells containing PMEs, indicating that pectin was demethylated by the PMEs from eight different plants. When recombinant PMEI was incubated with PMEs, stained zones disappeared. The results indicated that recombinant PMEI could inhibit the activities of PMEs from tomato, orange, carrot, apple, pineapple, hawthorn, grape and kiwi (Fig. 2).
Effects of pH and temperature on recombinant PMEI activity Excess amount of tomato fruit PME crude extract was mixed with refolded recombinant PMEI and the mixtures were pre-incu bated for 15 min (22.5 °C) at different pH ranging from 3 to10. Meanwhile, the mixtures were pre-incubated for 15 min (pH 7.0) at different temperature ranging from 20 to 60 °C. Residual PME activity was measured by continuous spectrophotometric assay, recording the titration of carboxyl groups released from a pectin solution with 0.01 M NaOH by an automatic pH-stat (Metrohm, Switzerland). Routine assays were performed with a 3.0 mg/mL citrus pectin (DE 70%, 20 ml) containing 0.117 M NaCl [14–16]. The inhibitory capacity was determined as pectinesterase-inhibitory percentage (%) = 100% ¡ residual PME activity (%) .
Fig. 1. SDS–PAGE analysis of PMEI expression and purification in E. coli. Lane1, pro tein molecular weight marker; lane 2, the purified inclusion bodies by the nickel ion metal chelating affinity chromatography; lane 3, the uninduced soluble frac tion of E. coli BL21 with pET30a-PMEI; lane 4, the induced soluble fraction of E. coli BL21 with pET30a-PMEI; lane 5, the uninduced insoluble fraction of E. coli BL21with pET30a-PMEI; lane 6, the induced insoluble fraction of E. coli BL21 with pET30a-PMEI.
Y. Hao et al. / Protein Expression and Purification 60 (2008) 221–224
Fig. 2. The inhibitory capacity of recombinant PMEI against various PMEs using gel diffusion assay. The wells were loaded with: 1, orange fruit PME; 2, orange fruit PME mixed with recombinant PMEI; 3, tomato fruit PME mixed with recombinant PMEI; 4, tomato fruit PME; 5, grape fruit PME mixed with recombinant PMEI; 6, grape fruit PME; 7, hawthorn fruit PME mixed with recombinant PMEI; 8, hawthorn fruit PME; 9, carrot fruit PME; 10, carrot fruit PME mixed with recombinant PMEI; 11, kiwi fruit PME; 12, kiwi fruit PME mixed with recombinant PMEI; 13, apple fruit PME; 14, apple fruit PME mixed with recombinant PMEI; 15, pineapple fruit PME; 16, pineapple fruit PME mixed with recombinant PMEI.
Effects of pH and temperature on recombinant PMEI activity
The recombinant PMEI inhibitory capacity to tomato PME was monitored at different pH ranging from 3 to 10, the results showed that different pH values had no obvious influence on the stability of the PME–PMEI complexes. Meanwhile, the recombinant PMEI inhibitory capacity was monitored at different temperature from 20 to 60 °C. The results demonstrated that recombinant PMEI effi ciently inhibited PME between 20 and 40 °C, but the inhibitory capacity rapidly decreased from 40 to 60 °C. The results suggested that recombinant PMEI could efficiently overcome the “cloud loss” in fruit and vegetable juice storage at room temperature.
We are grateful to Prof. Xinghua Guo for technical help and dis cussion. The research was supported by a National High-Tech R&D Program Grant (No. 2006AA10Z317, No. 2007AA10Z354 and No. 2006BAB04A06) from Ministry of Science and Technology of the People’s Republic of China.
Discussion In terms of biological activity, the formation of inclusion bodies remains a significant barrier in the E. coli expression system, due to the arduous task of refolding the aggregated protein . In order to decrease the formation of inclusion bodies, the bacterial cultures were grown below 37 °C. This will provide sufficient time for protein refolding since the rates of transcription and transla tion will substantially decrease at lower temperature . So, in this study, BL21 (DE3) with pET30a-PMEI was first grown at 25 °C, but the PMEI activity in soluble fraction was extremely low (data not shown). The reason may be that PMEI contains five cysteines residue to form two disulfide bonds, which are essential for the PMEI secondary structure. Whereas the reducing environment in E. coli cytoplasm can not facilitate extensive disulfide bond forma tion , PMEI may not be produced in their correct conforma tion in the cytoplasm. In order to obtain a high-level production of PMEI with biological activity, the recombinant E. coli BL21 was grown at 37 °C to form inclusion bodies. Then the aggregated pro tein was correctly refolded in the buffer with glutathione redox system . As a prokaryotic expression system, E. coli can not perform the post-translational modifications that are often required for the functional characteristics of the eukaryotic proteins . Although the kiwi PMEI was found to be glycosylated , the N-glycosidase F digestion assay showed that glycidic portion was buried in the native protein . So, we attempted to choose E. coli expression system to produce PMEI. The results showed that N-linked glyco sylation of PMEI may not be essential for the functions of PMEI. In addition, the recombinant PMEI remained stable inhibitor activ ity to tomato PME at 20–40 °C (pH 7.0). The results suggested that the recombinant PMEI has a potential application in the fruit juice products industry.
References  D. Castaldo, A.L. Voi, A. Giovane, L. Quagliuolo, L. Servillo, C. Balestrieri, Inhi bition of pectin methyl esterase in fruit juices, Report XX, Symposium of the International Fruit Juice Producers, Paris, France, 1990, pp. 333–338.  C. Balestrieri, D. Castaldo, A. Giovane, L. Quagliuolo, L. Servillo, A glycoprotein inhibitor of pectin methylesterase in kiwi fruit (Actinidia chinensis), Eur. J. Bio chem. 193 (1990) 183–187.  L. Camardella, V. Carratore, M.A. Ciardiello, L. Servillo, C. Balestrieri, A. Giov ane, Kiwi protein inhibitor of pectin methylesterase: amino-acid sequence and structural importance of two disulfide bridges, Eur. J. Biochem. 267 (2000) 4561–4565.  A.D. Matteo, A. Giovane, A. Raiola, L. Camardella, D. Bonivento, G.D. Lorenzo, F. Cervone, D. Bellincampi, D. Tsernoglou, Structural basis for the interaction between pectin methylesterase and a specific inhibitor protein, Plant Cell 17 (2005) 849–858.  A. Giovane, L. Servillo, A. Balestrieri, A. Raiola, D. Avino, M. Tamburrini, M.A. Ciardiello, L. Camardella, Pectin methylesterase inhibitor, Biochim. Biophys. Acta 1696 (2004) 245–252.  D. Castaldo, A. Lovoi, L. Quagliuolo, C. Balestrieri, A. Giovane, Orange juice and concentrates stabilization by a protein inhibitor of pectin methylesterase, J. Food Sci. 56 (1991) 1632–1633.  A. Giovane, B. Laratta, R. Loiudice, L. Quagliuolo, D. Castaldo, L. Servillo, Deter mination of residual pectin methylesterase activity in food products, Biotech nol. Appl. Biochem. 23 (1996) 181–184.  D. Casaldo, B. Laratta, R. Loiudice, A. Giovane, L. Quagliuolo, L. Servillo, Pres ence of residual pectin methylesterase activity in thermally stabilized indus trial fruit preparations, Lebensm. Wiss. U. Technol. 30 (1997) 479–484.  J.C. Yin, G.X. Li, X.F. Ren, G. Herrler, Select what you need: A comparative evalu ation of the advantages and limitations of frequently used expression systems for foreign genes, J. Biotechnol. 127 (2007) 335–347.  X.H. Mei, Y.L. Hao, H.L. Zhu, H.Y. Gao, Y.B. Luo, Cloning of pectin methyleserase inhibitor from kiwi fruit and its high expression in Pichia pastoris, Enzyme. Microb. Tech. 40 (2007) 1001–1005.  L. Tian, W.F. Kong, Q.H. Pan, J.C. Zhan, P.F. Wen, J.Y. Chen, S.B. Wan, W.D. Huang, Expression of the chalcone synthase gene from grape and preparation of an anti-CHS antibody, Protein Expr. Purif. 50 (2006) 223–228.  A. Giovane, L. Quagliuolo, L. Servillo, C. Balestrieri, B. Laratta, R. Loiudice, Puri fication and characterization of three isozymes of pectin methylesterase from tomato fruit, J. Food Biochem. 17 (1994) 339–349.  B. Downie, L.M.A. Dirk, K.A. Hadfield, T.A. Wilkins, A.B. Bennett, K.J. Bradford, A gel diffusion assay for quantification of pectin methylesterase activity, Anal. Biochem. 264 (1998) 149–157.  A.E. Hagerman, P.J. Austin, Continuous spectrophotometeric assay for plant pectin methyl esterase, J. Agric. Food Chem. 34 (1986) 440–444.  L.B. Nguyen, A.M.V. Loey, C. Smout, I. Verlent, T. Duvetter, M.E. Hendrickx, Effect of intrinsic and extrinsic factors on the interaction of plant pectin methy lesterase and its proteinaceous inhibitor from kiwi fruit, J. Agric. Food Chem. 52 (2004) 8144–8150.
Y. Hao et al. / Protein Expression and Purification 60 (2008) 221–224
 C.S. Nunes, S.M. Castro, J.A. Saraiva, M.A. Coimbra, M.E. Hendrickx, A.M. Vanloey, Thermal and high-pressure stability of purified pectin methylest erase from plums (prunus domestica), J. Food Biochem. 30 (2006) 138– 154.  C.M. Jiang, C.P. Li, H.M. Chang, Influence of pectinesterase inhibitor from jelly fig (ficus awkeotsang makino) achenes on pectinesterases and cloud loss of fruit juices, J. Food Sci. 8 (2002) 3063–3068.  R. Rudolph, G.N. Bennett, In vitro folding of inclusion body proteins, FASEB J. 10 (1996) 49–56.  S.C. Makrides, Strategies for achieving high-level expression of gene in Esche richia coli, Microbiol. R. 9 (1996) 512–538.
 C. Hwang, A.J. Sinskey, H.F. Lodish, Oxidized redox state of glutathione in the endoplasmic reticulum, Science 257 (1992) 1496–1502.  H. Wang, J.X. Dai, B.H. Li, K.X. Fan, L. Peng, D.P. Zhang, Z.G. Cao, W.Z. Qian, H. Wang, J. Zhao, Y.J. Guo, Expression, purification, and characterization of an immunotoxin containing a humanized anti-CD25 single-chain fragment vari able antibody fused to a modified truncated Pseudomonas exotoxin A, Protein Expr. Purif. 58 (2008) 40–147.  A. Giovane, C. Balestrieri, L. Quagliuolo, D. Castaldo, L. Servillo, A glycoprotein inhibitor of pectin methylesterase in kiwi fruit purification by affinity chroma tography and evidence of a ripening-related precursor, Eur. J. Biochem. 233 (1995) 926–929.