Protein Expression and Purification 94 (2014) 95–100
Contents lists available at ScienceDirect
Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep
Extracellular expression and efficient purification of a functional recombinant Volvariella volvacea immunomodulatory protein (FIP-vvo) using Pichia pastoris system Xilin Sun, Wei Huang, Sijia Xiao, Chongyang Liang, Shuqin Zhang, Zhiyi Liu, Fei Sun ⇑ Institute of Frontier Medical Science of Jilin University, Changchun City 130021, China
a r t i c l e
i n f o
Article history: Received 21 July 2013 and in revised form 25 October 2013 Available online 18 November 2013 Keywords: reFIP-vvo Volvariella volvacea Pichia pastoris Immunomodulatory
a b s t r a c t The fungal immunomodulatory proteins (FIPs) are a new protein family identified from several edible and medical mushrooms and play an important role in antitumor, anti-allergy and immunomodulating activities. A gene encoding the FIP-vvo was cloned from the mycelia of Volvariella volvacea and recombinant expressed in the Pichia pastoris expression system. SDS–PAGE, amino acid composition and circular dichroism analyses of the recombinant FIP-vvo (reFIP-vvo) indicated that the gene was correctly and successfully expressed. In vitro assays of biological activities revealed that the reFIP-vvo exhibited similar immunomodulating capacities as native form. The reFIP-vvo significantly stimulated the proliferation of mouse spleen lymphocytes and apparently enhanced the expression level of IFN-c released from the mouse splenocytes. Taken together, the FIP-vvo gene from V. volvacea has been integrated into the yeast genome and expressed effectively at a high level (about 410 mg/L), it was capable of agglutinating sheep and rat red blood cells. The reFIP-vvo possessed very similar biological activities to native FIPs, suggesting its potential application as a food supplement or immunomodulating agent in pharmaceuticals and even medical studies. Ó 2013 Elsevier Inc. All rights reserved.
Introduction Mushrooms have been used as the most common used medicine for thousands of years in Asia [1]. Fungal immunomodulatory proteins (FIPs)1 are a family of bioactive small molecular weight proteins, isolated from medicinal or edible mushrooms. Up to now, at least nine FIPs were identified, including LZ-8, FIP-gts, FIP-fve, FIP-vvo, FIP-gsi, etc., which are from Ganoderma lucidum [2], Ganoderma tsugae [3], Flammulina velutipes [4], Volvariella volvacea [5], and Ganoderma sinense [6] respectively. These FIPs consist of 110–114 amino acid residues with a molecular weight of approximately 13 kDa [7], amino acid sequences of FIPs show a 60–100% similarity. These FIPs resemble lectins or immunoglobulins in terms of structure and immunomodulating activities [7,8]. FIPs are mitogenic in vitro for human peripheral blood lymphocytes (hPBLs) and mouse splenocytes, and induce a bell-shaped dose–response curve similar to that for lectin mitogens [3]. FIP-vvo was shown to stimulate the maximum proliferation of human peripheral blood lymphocytes at a
⇑ Corresponding author. Tel.: +86 431 85622960. E-mail address: [email protected] (F. Sun). Abbreviations used: FIPs, fungal immunomodulatory proteins; HRP, horseradish peroxidase 1
1046-5928/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pep.2013.10.022
concentration of 5 lg/mL, and it could agglutinate red blood cells of rat and rabbit [5]. Only 115 mg of FIP-vvo was purified from 3 kg fresh fruit bodies of V. volvacea after all purification steps [5,7]. Approximately 15–30 mg of purified LZ-8 (G. lucidum immunomodulatory protein) was obtained from 1 kg wet G. lucidum mycelia [2]. Thus, isolating natural FIPs from fungus is an overly time-consume and costly work. Therefore, a plenty of researches were focusing in heterologous expression and purification of FIPs last decade [9–11]. So far, heterologous expression of FIP-vvo has been reported using Escherichia coli [11]. However, E. coli is a prokaryote and lacks intracellular organelles, such as the endoplasmic reticulum and the golgi apparatus that are present in eukaryotes, which are responsible for modifications of the proteins being produced. Many eukaryotic proteins can be produced in E. coli but are produced in a nonfunctional, unfinished form, since glycosylation or post-translational modifications do not occur. Therefore, researchers have recently turned to eukaryotic yeast and mammalian expression systems for protein production [12]. Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and easy way [13]. In this study, we established an efficient method to produce and purify large quantities of recombinant FIP-vvo (reFIP-vvo) in bioactive form using P. pastoris.
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Fig. 1. (A) Plasmid structure of pPICZaA-FIP-vvo. (B) PCR analysis of Pichia integrants. (C) Western blot analysis of the supernatant of PCR positive clone, with anti-FIP-vvo antibody. 1, 2, 3, 4, 5 Supernatant of fermentation broth from Pichia pastoris containing a pPICZaA-FIP-vvo after induction with methanol; 6 supernatant from Pichia pastoris containing a pPICZaA empty vector after induction with methanol. The numeration of the lanes is the same as in (B).
Fig. 2. SDS–PAGE of fermentation and Purification of reFIP-vvo. (A) M, protein marker; supernatant of fermentation broth after expression for 18 h (lane 1), 24 h (lane 2), 30 h (lane 3), 36 h (lane 4) and 42 h (lane 5), respectively. (B) SDS–PAGE analysis of the fractions collected during purification: M, protein marker; 3, sample after filtration and dialyze; 2, eluate with elution buffer from Hiload 26/20 SP Sepharose XL column Cation exchange resin; 1, protein eluted with 50 mM Tris–HCl from Hiload 16/60 Superdex 75 column.
Materials and methods Reagents EcoRI and KpnI were purchased from New England Biolabs (USA). FastAP and T4 DNA ligase was purchased from Fermentas (Canada). Zeocine was purchased from Invitrogen (CA, USA). Rabbit
anti-FIP-vvo polyclonal antibody was purchased from GL Biochem (Shanghai, China). Horseradish peroxidase (HRP) labeled goat antirabbit IgG was purchased from Santa (USA). DNA markers and protein markers were purchased from TIANGEN (Beijing, China). The other reagents used in this study were of analytical grade and were commercially available. The culture media, YPD, BMGY and BMMY were prepared according to the Invitrogen manufacturer’s guidelines.
X. Sun et al. / Protein Expression and Purification 94 (2014) 95–100
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Transformation of P. pastoris and screening for transformants
Table 1 Purification summary of reFIP-vvo from culture medium of P. pastoris. Purification step
Total proteina (mg)
Purity (%)
reFIP-vvo (mg)
Yieldb (%)
Culture supernatant (400 ml) Cation exchange Gel permeation
646.8
84.1
544
100
96.4 54.9
91.7 95
88.4 52.1
16.0 9.6
a
Wet weight (152.5 g) yeast cells from 500 ml culture were harvested. Protein yield was calculated using the amount of reFIP-vvo after concentration and reFIP-vvo amount in culture supernatant. b
Strains and vectors P. pastoris strain X-33 is a wild-type Pichia strain that is useful for selection on Zeocin™ and large scale growth. X-33 and plasmid pPICZaA (3329 bp) were purchased from Invitrogen (CA, USA). Recombinant vectors pPICZaA-FIP-vvo were constructed in our laboratory.
Oligonucleotides One set of primers was designed to amplify the FIP-vvo gene. Primers used for PCR were synthesized by Sangon Biotechnology Corp. (Shanghai, China).
Shadow indicates site for KpnI sequence, double underline indicates site for EcoRI sequence. The PCR was performed with Takara Primestar DNA Polymerase.
Construction of expression vector Based on the amino acid sequence of wild FIP-vvo published before [5], a DNA sequence(339 bp) encoding the recombinant FIP-vvo (reFIP-vvo) was designed using the Pichia pastoris preferred codons (http://www.kazusa.or.jp/codon/) and was synthesized (in the vector pUCE) by Inovogen Tech. Corp. (Beijing, China). Restriction enzyme cutting site (EcoRI and KpnI) was added by PCR (Primer pairs: reFIP-vvo-F; reFIP-vvo-R) in order to insert into the pPICZaA vector in its MSC. Furthermore, the PCR product was digested with KpnI and EcoRI and was ligated into the same enzyme digested vector pPICZaA, resulting in the recombinant vector pPICZaA–FIP-vvo. The recombinant vector was transformed into E. coli DH5a for amplification and DNA sequence analysis.
Recombinant vector pPICZaA-FIP-vvo (10 ng) was linearized by PmeI (New England Biolabs, USA) and introduced into P. pastoris X-33 by electroporation. Five micro-liter of YPD medium was inoculated with 50 ll of cryopreserved culture and cultivated overnight at 30 °C. 250 ml of YPD medium in 1L conical flask was inoculated with 50 ll of the night culture and cultivated at 30 °C until OD600 reached 1.3–1.5. The culture was centrifuged (4000g, 10 min, 4 °C) and the pellet was resuspended in 500 ml of ice-cold sterile water. The cells were centrifuged as described above. The pellet was resuspended in 250 ml of ice-cold sterile water and centrifuged. Then the pellet was resuspended in 20 ml of ice-cold 1 M sorbitol and centrifuged. Finally the pellet was resuspended in 1 ml of ice-cold 1 M sorbitol. Such prepared competent P. pastoris cells were transformed with the linearized expression vector pPICZaA-FIP-vvo by electroporation according to the manufacturer’s instructions. Transformants were initially screened on YPDS plate (Containing 100 ng/ml Zeocin) for their resistance to Zeocin. The positive recombinants were identified by PCR using primer pairs (fip-vvo-up; fip-vvo-down) and PCR products were analyzed on a 1% agrose gel. The recombinants were inoculated in 5 ml YPD medium at 28 °C overnight, then 20 ll culture were transferred to 20 ml BMGY medium and was incubated at 28 °C overnight. The cells were then collected by centrifugation at 5000g for 10 min and resuspended in 20 ml BMMY medium. The cultures were incubated for 96 h at 28 °C with constant shaking, and 100% methanol was added to a final concentration of 0.5% every 24 h to maintain induction. The supernatant was harvested by centrifugation at 15000g for 3 min. Coomassie-stained SDS–PAGE and Western blot Fifteen percent SDS–PAGE was performed, and the gel was stained with Coomassie brilliant blue R250. For Western Blot, after separation by SDS–PAGE, proteins were transferred onto a PVDF membrane. The membrane was blocked with 5% dried skim milk in TBST (TBS containing 0.1% Tween 20) overnight at 4 °C and incubated with rabbit anti-FIP-vvo polyclonal antibody (1:3000) for 2 h at 25 °C. The membrane was washed six times with TBST and incubated with horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1:8000) for 2 h at 25 °C. Finally, the membrane was washed again with TBST and developed with diaminobenzidine (DAB) as the substrate. Large-scale expression For the large scale production of recombinant FIP-vvo the same procedure was applied, using 1 L of BMGY medium in 3 L flask for
Fig. 3. The results of secondary structure and molecular weight of reFIP-vvo characterized by CD (A) and MS (B).
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Fig. 4. Hemagglutination activity of reFIP-vvo. (A) Results of Wistar rats red blood cells. Lane 1: saline, lane 14: ConA (5 lg/mL), lane 2–13: diluted reFIP-vvo (0.1 lg/mL, 0.2 lg/mL, 0.3 lg/mL, 0.4 lg/mL, 0.5 lg/mL, 0.75 lg/mL, 1 lg/mL, 1.25 lg/mL, 1.5 lg/mL, 3 lg/mL, 10 lg/mL, 50 lg/mL); (B) Results of sheep red blood cells. Lane 1: saline, lane 16: ConA (5 lg/mL), lane 2–15: diluted reFIP-vvo (0.5 lg/mL, 1 lg/mL, 1.1 lg/mL, 1.2 lg/mL, 1.3 lg/mL, 1.4 lg/mL, 1.5 lg/mL, 2 lg/mL, 2.5 lg/mL, 3 lg/mL, 4 lg/mL, 5 lg/mL, 10 lg/mL, 50 lg/mL).
overnight preculture. The cells were then collected and resuspended in 200 ml of BMMY medium in 1 L flask and incubated at 28 °C on a rotary shaker with methanol induction (0.5% v/v) every 24 h for three days. A 10 ml culture of the best clone was inoculated into 300 mL BMGY of medium, which was then incubated with aeration at 28 °C until the cell density reached OD600 = 10. Then, the shaken starter flask culture was inoculated into an 7.5 L NBS BioFlo bioreactor (New Brunswick Scientific, USA) containing 3.5 L of medium. The protocol for the fermentation process, including the feed rates and the phases of fermentation, was performed as described in the Invitrogen Manual for the P. pastoris Fermentation Process. The glycerol feed was used until a cell yield of 180–220 g/L wet weight was achieved. Methanol at a final concentration of 0.5% was then used as the carbon source to induce the AOX1 promoter. The culture was finally harvested after being fed on methanol for 48 h. Specimens from the culture supernatant were analyzed to determine the amount of cell growth (OD600 and wet cell weight), the pH, the microscopic purity and the protein concentrations. Meanwhile, during the fermentation process, samples were taken every 6 h to monitor the expression levels of the reFIP-vvo.
Fig. 5. The effect of other mitogens (Con A) on the stimulation of mouse spleen lymphocytes by reFIP-vvo. Dose–response of spleen cells to reFIP-vvo and/or Con A. Blast-formation stimulatory activity of spleen cells was performed as described under ‘‘Materials and methods’’ in the presence of reFIP-vvo (0.3 lg/mL, 0.4 lg/mL, 0.5 lg/mL, 1 lg/mL, 2 lg/mL, 3 lg/mL, 4 lg/mL, 5 lg/mL), PBS, Con A, p < 0.05.
Purification of reFIP-vvo The cells were removed from supernatant by centrifugation (3000g, 10 min, 20 °C) and filtration with a 0.22 lm filter. After filtration approximately 500 mL supernatant was dialyzed against water for two days and its pH was adjusted to 5.5. The reFIP-vvo was purified in a single step by cation exchange chromatography, Hiload 26/20 SP Sepharose XL column (Amersham Biosciences), 20 mM sodium citrate–citrate buffer pH5.5, linear gradient 0–1 M NaCl) using ÄKTA Purifier protein chromatography system (Amersham Biosciences, GE), followed by gel permeation chromatography, Hiload 16/60 Superdex 75 column (Amersham Biosciences, GE), 50 mM Tris–HCl, pH 8.0 using ÄKTA Purifier protein chromatography system (Amersham Biosciences, GE). The purity of the pooled fractions was checked by 15% SDS–PAGE, the concentration of proteins was determined using Kjeldahl
determination. MALDI-TOF-MS and Circular Dichroism (CD) were measured by Beijing Proteome Research center. Haemagglutination assay Haemagglutination activity was measured as described previously [2]. Fresh blood was obtained from Wistar rats and sheep stored in a specific pyrogen-free room. After washing 6 ml of whole blood with physiological saline solution three times, blood cells were collected by centrifugation at 500g for 10 min and then suspended to 1.0% (v/v) with physiological saline solution. Suspensions of 100 lL of purified reFIP-vvo (final concentration from 0.1 lg/mL to 5 lg/mL) in physiological saline solution, and 100 lL of 1.0% red blood cells placed in 96-well microtiter plates (round bottom, Nunc)
X. Sun et al. / Protein Expression and Purification 94 (2014) 95–100
were shaken for 30 s at room temperature and then incubated at 37 °C. The plates were examined for haemagglutination after 2 h. Mouse splenocytes proliferation assay Female BALB/c mice aged 8–12 weeks were maintained under standard pathogen-free conditions. Murine splenocytes were prepared as previously described [14] with minor modifications. Mouse spleens were surgically removed and minced at room temperature in RPMI 1640 medium. The tissue debris was removed by passing the cell suspension through a cotton column. Splenocytes (2 105 per well) were seeded in a 96-well plate, with a volume of 100 lL. Then 100 lL purified reFIP-vvo diluted in medium were added at the final concentrations of 0.3–5 lg/mL, incubated for 24 h. Finally 20 ll WST-1 was added to each well and incubated at 37 °C for another 2.5 h, and then the mouse splenocytes proliferation were quantified by measuring the absorbance at 450 nm. ConA (5 lg/mL) and medium with 1% PBS were applied as positive and negative controls, respectively. Results and discussion Expression of reFIP-vvo in X-33 The coding gene for reFIP-vvo was designed according to the codon usage of P. pastoris and was amplified by PCR using primers mentioned above from a synthesized vector bearing the DNA sequence of reFIP-vvo. To create the expression construct of pPICZaA-FIP-vvo (Fig. 1A), amplified gene of reFIP-vvo was cloned into the pPICZaA expression vector via the 50 EcoRI and 30 KpnI restriction sites. The pPICZaA bearing the DNA sequence of reFIP-vvo was linearized and transformed in the P. pastoris X-33 strain by electroporation. In the pPICZaA-FIP-vvo, the FIP-vvo gene was under the control of the AOX1 promoter allowed for the protein secretion in P. pastoris by induction with methanol and using the yeast signal a-factor included in the vector for extracellular targeting of the expressed protein. P. pastoris was cultivated on nutrient rich (BMGY/BMMY) media recommended by the supplier (Invitrogen, USA) for three days. Genomic DNA was isolated from clones to determine if the gene of interest has integrated into the Pichia genome. We amplify the gene of interest by PCR and analyzed on a 1% agarose gel (Fig. 1B). The supernatant of the clones with positive result in the PCR analysis were tested by western blot analysis (Fig. 1C). reFIP-vvo was expressed in culture media used and the yield of reFIP-vvo as shown in Fig. 2A increased upon induction by methanol at time point of 18, 24, 30, 36 and 42 h. Moreover, to the best of our knowledge, this is the first successful heterologous expression and production of FIP-vvo in a Pichia expression system to be reported.
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even more favorable. reFIP-vvo was obtained in a high yield and amount (Table 1). The purity of the reFIP-vvo was confirmed by SDS–PAGE analysis, showing a single band of 12 kDa, which is the same as for the wild type FIP-vvo. Initial expression experiments of transformed P. pastoris cells showed that the band of reFIP-vvo appeared at approx. 12 kDa in SDS polyacrylamide gel. The results of MALDI-TOF-MS confirmed the molecular weight of reFIP-vvo was approximately 12,658.20 Da (Fig. 3B), which correspond with theoretical Mr.12667.23 Da (http://expasy.org/cgi-bin/pi_tool). As described in the Methods, reFIP-vvo was purified to homogeneity in only one step as seen in Table 1. An overall concentration of 3.84 mg/ mL was achieved. As shown in Fig. 3A, the percentage of secondary structures in reFIP-vvo are 16.8% (a-helix), 31.8% (b-sheet), 16.8% (a-helix), 19.2% (b-turn), 32.2% (Random). Bioactivity of reFIP-vvo In vitro, the reFIP-vvo was capable of hemagglutinating red blood cells of Wistar rats (at concentrations greater than 0.2 lg/ml) and sheep (at concentrations greater than 1.0 lg/ml), but no such activity was observed toward human red blood cells, which were similar to the native FIP-vvo (Fig. 4). reFIP-vvo also plays a role as a mitogen in mouse spleen cells. The reFIP-vvo enhanced the proliferation of mouse spleen cells (Fig. 5). The mouse splenocyte proliferation activity of reFIP-vvo expressed in P. pastoris was much higher than the reFIP-vvo expressed in E. coli [15]. Now that the FIP-vvo gene has been expressed in the P. pastoris system, but more work needs to be done in improving the expression level and scaling-up to industrial production. The anti-tumor and anti-allergic activities in vivo also need to be further demonstrated. Additionally, the mechanism of how the FIP-vvo exert its effects on immune cells needs to be investigated and elucidated. Nevertheless, this study might supply some basis for the largescale production of recombinant FIP-vvo and its potential application in oral administration or therapeutic utilization in medical researches. Conclusions Here FIP-vvo gene from V. volvacea has been integrated into the yeast genome and expressed effectively at a high level (about 500 mg/L). The reFIP-vvo possessed very similar biological activities to native FIP-vvo, suggesting its potential application as a food supplement or immunomodulating agent in pharmaceuticals and even medical studies. References
Purification of the reFIP-vvo reFIP-vvo was purified from the culture medium of X-33 after three days of cultivation as described above. After filtration with a 0.22 lm filter, approximately 400 mL supernatant was dialyzed against water for 2 days and was loaded to the Hiload 26/20 SP Sepharose XL column. And purified further by Hiload 16/60 Superdex 75 column (Amersham Biosciences). The purity of the pooled fractions was checked by SDS–PAGE (Fig. 2B). The protein sample was concentrated to 3.8 mg/mL in the buffer of 50 mM NaCl, 20 mM Tris–HCl, pH 7.0 for further use. The isoelectric point was experimentally determined to be 7.83. Extracellular reFIP-vvo secreted by P. pastoris into medium was purified to homogeneity by a single cation exchange chromatography step, thus making the recombinant production of the protein
[1] X. Xu, H. Yan, J. Chen, X. Zhang, Bioactive proteins from mushrooms, Biotechnol. Adv. 29 (2011) 667–674. [2] K. Kino, A. Yamashita, K. Yamaoka, J. Watanabe, S. Tanaka, K. Ko, K. Shimizu, H. Tsunoo, Isolation and characterization of a new immunomodulatory protein, ling zhi-8 (LZ-8), from Ganoderma lucidium, J. Biol. Chem. 264 (1989) 472–478. [3] W.H. Lin, C.H. Hung, C.I. Hsu, J.Y. Lin, Dimerization of the N-terminal amphipathic a-Helix Domain of the fungal immunomodulatory protein from Ganoderma tsugae (Fip-gts) defined by a yeast two-hybrid system and sitedirected mutagenesis, J. Biol. Chem. 272 (1997) 20044–20048. [4] J.L. Ko, S.J. Lin, C.I. Hsu, C.L. Kao, J.Y. Lin, Molecular cloning and expression of a fungal immunomodulatory protein, FIP-fve, from Flammulina velutipes, J. Formos. Med. Assoc. 96 (1997) 517–524. [5] H.C. Hsu, C.I. Hsu, R.H. Lin, C.L. Kao, J.Y. Lin, Fip-vvo, a new fungal immunomodulatory protein isolated from Volvariella volvacea, Biochem. J. 323 (1997) 557–565. [6] X.W. Zhou, M.Q. Xie, F. Hong, Q.Z. Li, Genomic cloning and characterization of a FIP-gsi gene encoding a fungal immunomodulatory protein from Ganoderma sinense Zhao et al. (Aphyllophoromycetideae), Int. J. Med. Mushrooms 11 (2009) 77–86.
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[7] F. Han, Y. Liu, L.Q. Guo, X.L. Zeng, Z.M. Liu, J.F. Lin, Heterologous expression of the immunomodulatory protein gene from Ganoderma sinense in the basidiomycete Coprinopsis cinerea, J. Appl. Microbiol. 109 (2010) 1838– 1844. [8] R.S. Singh, R. Bhari, H.P. Kaur, Mushroom lectins: current status and future perspectives, Crit. Rev. Biotechnol. 30 (2010) 99–126. [9] C. Liang, S. Zhang, Z. Liu, F. Sun, Ganoderma lucidum immunomodulatory protein (Lz-8) expressed in Pichia pastoris and the identification of immunocompetence, Sheng Wu Gong Cheng Xue Bao 25 (2009) 441– 447. [10] C.M. Wu, T.Y. Wu, S.S. Kao, J.L. Ko, T.R. Jinn, Expression and purification of a recombinant Fip-fve protein from Flammulina velutipes in baculovirus-infected insect cells, J. Appl. Microbiol. 104 (2008) 1354– 1362.
[11] X.F. Wang, Q.Z. Li, T.W. Bao, W.R. Cong, W.X. Song, X.W. Zhou, In vitro rapid evolution of fungal immunomodulatory proteins by DNA family shuffling, Appl. Microbiol. Biotechnol. (2012) 2455–2465. [12] S. Macauley-Patrick, M.L. Fazenda, B. McNeil, L.M. Harvey, Heterologous protein production using the Pichia pastoris expression system, Yeast 22 (2005) 249–270. [13] M. Weidner, M. Taupp, S.J. Hallam, Expression of recombinant proteins in the methylotrophic yeast Pichia pastoris, J. Vis. Exp. (2010) 1–5. [14] L. Sommese, G. Donnarumma, C. de l’Ero, A. Marcatili, M. Vitiello, M. Galdiero, Growth hormone modulates IL-alpha and IFN-gamma release by murine splenocytes activated by LPS or porins of Salmonella typhimurium, J. Med. Microbiol. 45 (1996) 40–47. [15] X.W. Zhou, Gene sequence of encoding fungal immunomodulatory protein of straw mushroom, China, CN 200910047782, August 19, 2009.
Contents lists available at ScienceDirect
Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep
Extracellular expression and efficient purification of a functional recombinant Volvariella volvacea immunomodulatory protein (FIP-vvo) using Pichia pastoris system Xilin Sun, Wei Huang, Sijia Xiao, Chongyang Liang, Shuqin Zhang, Zhiyi Liu, Fei Sun ⇑ Institute of Frontier Medical Science of Jilin University, Changchun City 130021, China
a r t i c l e
i n f o
Article history: Received 21 July 2013 and in revised form 25 October 2013 Available online 18 November 2013 Keywords: reFIP-vvo Volvariella volvacea Pichia pastoris Immunomodulatory
a b s t r a c t The fungal immunomodulatory proteins (FIPs) are a new protein family identified from several edible and medical mushrooms and play an important role in antitumor, anti-allergy and immunomodulating activities. A gene encoding the FIP-vvo was cloned from the mycelia of Volvariella volvacea and recombinant expressed in the Pichia pastoris expression system. SDS–PAGE, amino acid composition and circular dichroism analyses of the recombinant FIP-vvo (reFIP-vvo) indicated that the gene was correctly and successfully expressed. In vitro assays of biological activities revealed that the reFIP-vvo exhibited similar immunomodulating capacities as native form. The reFIP-vvo significantly stimulated the proliferation of mouse spleen lymphocytes and apparently enhanced the expression level of IFN-c released from the mouse splenocytes. Taken together, the FIP-vvo gene from V. volvacea has been integrated into the yeast genome and expressed effectively at a high level (about 410 mg/L), it was capable of agglutinating sheep and rat red blood cells. The reFIP-vvo possessed very similar biological activities to native FIPs, suggesting its potential application as a food supplement or immunomodulating agent in pharmaceuticals and even medical studies. Ó 2013 Elsevier Inc. All rights reserved.
Introduction Mushrooms have been used as the most common used medicine for thousands of years in Asia [1]. Fungal immunomodulatory proteins (FIPs)1 are a family of bioactive small molecular weight proteins, isolated from medicinal or edible mushrooms. Up to now, at least nine FIPs were identified, including LZ-8, FIP-gts, FIP-fve, FIP-vvo, FIP-gsi, etc., which are from Ganoderma lucidum [2], Ganoderma tsugae [3], Flammulina velutipes [4], Volvariella volvacea [5], and Ganoderma sinense [6] respectively. These FIPs consist of 110–114 amino acid residues with a molecular weight of approximately 13 kDa [7], amino acid sequences of FIPs show a 60–100% similarity. These FIPs resemble lectins or immunoglobulins in terms of structure and immunomodulating activities [7,8]. FIPs are mitogenic in vitro for human peripheral blood lymphocytes (hPBLs) and mouse splenocytes, and induce a bell-shaped dose–response curve similar to that for lectin mitogens [3]. FIP-vvo was shown to stimulate the maximum proliferation of human peripheral blood lymphocytes at a
⇑ Corresponding author. Tel.: +86 431 85622960. E-mail address: [email protected] (F. Sun). Abbreviations used: FIPs, fungal immunomodulatory proteins; HRP, horseradish peroxidase 1
1046-5928/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pep.2013.10.022
concentration of 5 lg/mL, and it could agglutinate red blood cells of rat and rabbit [5]. Only 115 mg of FIP-vvo was purified from 3 kg fresh fruit bodies of V. volvacea after all purification steps [5,7]. Approximately 15–30 mg of purified LZ-8 (G. lucidum immunomodulatory protein) was obtained from 1 kg wet G. lucidum mycelia [2]. Thus, isolating natural FIPs from fungus is an overly time-consume and costly work. Therefore, a plenty of researches were focusing in heterologous expression and purification of FIPs last decade [9–11]. So far, heterologous expression of FIP-vvo has been reported using Escherichia coli [11]. However, E. coli is a prokaryote and lacks intracellular organelles, such as the endoplasmic reticulum and the golgi apparatus that are present in eukaryotes, which are responsible for modifications of the proteins being produced. Many eukaryotic proteins can be produced in E. coli but are produced in a nonfunctional, unfinished form, since glycosylation or post-translational modifications do not occur. Therefore, researchers have recently turned to eukaryotic yeast and mammalian expression systems for protein production [12]. Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and easy way [13]. In this study, we established an efficient method to produce and purify large quantities of recombinant FIP-vvo (reFIP-vvo) in bioactive form using P. pastoris.
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Fig. 1. (A) Plasmid structure of pPICZaA-FIP-vvo. (B) PCR analysis of Pichia integrants. (C) Western blot analysis of the supernatant of PCR positive clone, with anti-FIP-vvo antibody. 1, 2, 3, 4, 5 Supernatant of fermentation broth from Pichia pastoris containing a pPICZaA-FIP-vvo after induction with methanol; 6 supernatant from Pichia pastoris containing a pPICZaA empty vector after induction with methanol. The numeration of the lanes is the same as in (B).
Fig. 2. SDS–PAGE of fermentation and Purification of reFIP-vvo. (A) M, protein marker; supernatant of fermentation broth after expression for 18 h (lane 1), 24 h (lane 2), 30 h (lane 3), 36 h (lane 4) and 42 h (lane 5), respectively. (B) SDS–PAGE analysis of the fractions collected during purification: M, protein marker; 3, sample after filtration and dialyze; 2, eluate with elution buffer from Hiload 26/20 SP Sepharose XL column Cation exchange resin; 1, protein eluted with 50 mM Tris–HCl from Hiload 16/60 Superdex 75 column.
Materials and methods Reagents EcoRI and KpnI were purchased from New England Biolabs (USA). FastAP and T4 DNA ligase was purchased from Fermentas (Canada). Zeocine was purchased from Invitrogen (CA, USA). Rabbit
anti-FIP-vvo polyclonal antibody was purchased from GL Biochem (Shanghai, China). Horseradish peroxidase (HRP) labeled goat antirabbit IgG was purchased from Santa (USA). DNA markers and protein markers were purchased from TIANGEN (Beijing, China). The other reagents used in this study were of analytical grade and were commercially available. The culture media, YPD, BMGY and BMMY were prepared according to the Invitrogen manufacturer’s guidelines.
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Transformation of P. pastoris and screening for transformants
Table 1 Purification summary of reFIP-vvo from culture medium of P. pastoris. Purification step
Total proteina (mg)
Purity (%)
reFIP-vvo (mg)
Yieldb (%)
Culture supernatant (400 ml) Cation exchange Gel permeation
646.8
84.1
544
100
96.4 54.9
91.7 95
88.4 52.1
16.0 9.6
a
Wet weight (152.5 g) yeast cells from 500 ml culture were harvested. Protein yield was calculated using the amount of reFIP-vvo after concentration and reFIP-vvo amount in culture supernatant. b
Strains and vectors P. pastoris strain X-33 is a wild-type Pichia strain that is useful for selection on Zeocin™ and large scale growth. X-33 and plasmid pPICZaA (3329 bp) were purchased from Invitrogen (CA, USA). Recombinant vectors pPICZaA-FIP-vvo were constructed in our laboratory.
Oligonucleotides One set of primers was designed to amplify the FIP-vvo gene. Primers used for PCR were synthesized by Sangon Biotechnology Corp. (Shanghai, China).
Shadow indicates site for KpnI sequence, double underline indicates site for EcoRI sequence. The PCR was performed with Takara Primestar DNA Polymerase.
Construction of expression vector Based on the amino acid sequence of wild FIP-vvo published before [5], a DNA sequence(339 bp) encoding the recombinant FIP-vvo (reFIP-vvo) was designed using the Pichia pastoris preferred codons (http://www.kazusa.or.jp/codon/) and was synthesized (in the vector pUCE) by Inovogen Tech. Corp. (Beijing, China). Restriction enzyme cutting site (EcoRI and KpnI) was added by PCR (Primer pairs: reFIP-vvo-F; reFIP-vvo-R) in order to insert into the pPICZaA vector in its MSC. Furthermore, the PCR product was digested with KpnI and EcoRI and was ligated into the same enzyme digested vector pPICZaA, resulting in the recombinant vector pPICZaA–FIP-vvo. The recombinant vector was transformed into E. coli DH5a for amplification and DNA sequence analysis.
Recombinant vector pPICZaA-FIP-vvo (10 ng) was linearized by PmeI (New England Biolabs, USA) and introduced into P. pastoris X-33 by electroporation. Five micro-liter of YPD medium was inoculated with 50 ll of cryopreserved culture and cultivated overnight at 30 °C. 250 ml of YPD medium in 1L conical flask was inoculated with 50 ll of the night culture and cultivated at 30 °C until OD600 reached 1.3–1.5. The culture was centrifuged (4000g, 10 min, 4 °C) and the pellet was resuspended in 500 ml of ice-cold sterile water. The cells were centrifuged as described above. The pellet was resuspended in 250 ml of ice-cold sterile water and centrifuged. Then the pellet was resuspended in 20 ml of ice-cold 1 M sorbitol and centrifuged. Finally the pellet was resuspended in 1 ml of ice-cold 1 M sorbitol. Such prepared competent P. pastoris cells were transformed with the linearized expression vector pPICZaA-FIP-vvo by electroporation according to the manufacturer’s instructions. Transformants were initially screened on YPDS plate (Containing 100 ng/ml Zeocin) for their resistance to Zeocin. The positive recombinants were identified by PCR using primer pairs (fip-vvo-up; fip-vvo-down) and PCR products were analyzed on a 1% agrose gel. The recombinants were inoculated in 5 ml YPD medium at 28 °C overnight, then 20 ll culture were transferred to 20 ml BMGY medium and was incubated at 28 °C overnight. The cells were then collected by centrifugation at 5000g for 10 min and resuspended in 20 ml BMMY medium. The cultures were incubated for 96 h at 28 °C with constant shaking, and 100% methanol was added to a final concentration of 0.5% every 24 h to maintain induction. The supernatant was harvested by centrifugation at 15000g for 3 min. Coomassie-stained SDS–PAGE and Western blot Fifteen percent SDS–PAGE was performed, and the gel was stained with Coomassie brilliant blue R250. For Western Blot, after separation by SDS–PAGE, proteins were transferred onto a PVDF membrane. The membrane was blocked with 5% dried skim milk in TBST (TBS containing 0.1% Tween 20) overnight at 4 °C and incubated with rabbit anti-FIP-vvo polyclonal antibody (1:3000) for 2 h at 25 °C. The membrane was washed six times with TBST and incubated with horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1:8000) for 2 h at 25 °C. Finally, the membrane was washed again with TBST and developed with diaminobenzidine (DAB) as the substrate. Large-scale expression For the large scale production of recombinant FIP-vvo the same procedure was applied, using 1 L of BMGY medium in 3 L flask for
Fig. 3. The results of secondary structure and molecular weight of reFIP-vvo characterized by CD (A) and MS (B).
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Fig. 4. Hemagglutination activity of reFIP-vvo. (A) Results of Wistar rats red blood cells. Lane 1: saline, lane 14: ConA (5 lg/mL), lane 2–13: diluted reFIP-vvo (0.1 lg/mL, 0.2 lg/mL, 0.3 lg/mL, 0.4 lg/mL, 0.5 lg/mL, 0.75 lg/mL, 1 lg/mL, 1.25 lg/mL, 1.5 lg/mL, 3 lg/mL, 10 lg/mL, 50 lg/mL); (B) Results of sheep red blood cells. Lane 1: saline, lane 16: ConA (5 lg/mL), lane 2–15: diluted reFIP-vvo (0.5 lg/mL, 1 lg/mL, 1.1 lg/mL, 1.2 lg/mL, 1.3 lg/mL, 1.4 lg/mL, 1.5 lg/mL, 2 lg/mL, 2.5 lg/mL, 3 lg/mL, 4 lg/mL, 5 lg/mL, 10 lg/mL, 50 lg/mL).
overnight preculture. The cells were then collected and resuspended in 200 ml of BMMY medium in 1 L flask and incubated at 28 °C on a rotary shaker with methanol induction (0.5% v/v) every 24 h for three days. A 10 ml culture of the best clone was inoculated into 300 mL BMGY of medium, which was then incubated with aeration at 28 °C until the cell density reached OD600 = 10. Then, the shaken starter flask culture was inoculated into an 7.5 L NBS BioFlo bioreactor (New Brunswick Scientific, USA) containing 3.5 L of medium. The protocol for the fermentation process, including the feed rates and the phases of fermentation, was performed as described in the Invitrogen Manual for the P. pastoris Fermentation Process. The glycerol feed was used until a cell yield of 180–220 g/L wet weight was achieved. Methanol at a final concentration of 0.5% was then used as the carbon source to induce the AOX1 promoter. The culture was finally harvested after being fed on methanol for 48 h. Specimens from the culture supernatant were analyzed to determine the amount of cell growth (OD600 and wet cell weight), the pH, the microscopic purity and the protein concentrations. Meanwhile, during the fermentation process, samples were taken every 6 h to monitor the expression levels of the reFIP-vvo.
Fig. 5. The effect of other mitogens (Con A) on the stimulation of mouse spleen lymphocytes by reFIP-vvo. Dose–response of spleen cells to reFIP-vvo and/or Con A. Blast-formation stimulatory activity of spleen cells was performed as described under ‘‘Materials and methods’’ in the presence of reFIP-vvo (0.3 lg/mL, 0.4 lg/mL, 0.5 lg/mL, 1 lg/mL, 2 lg/mL, 3 lg/mL, 4 lg/mL, 5 lg/mL), PBS, Con A, p < 0.05.
Purification of reFIP-vvo The cells were removed from supernatant by centrifugation (3000g, 10 min, 20 °C) and filtration with a 0.22 lm filter. After filtration approximately 500 mL supernatant was dialyzed against water for two days and its pH was adjusted to 5.5. The reFIP-vvo was purified in a single step by cation exchange chromatography, Hiload 26/20 SP Sepharose XL column (Amersham Biosciences), 20 mM sodium citrate–citrate buffer pH5.5, linear gradient 0–1 M NaCl) using ÄKTA Purifier protein chromatography system (Amersham Biosciences, GE), followed by gel permeation chromatography, Hiload 16/60 Superdex 75 column (Amersham Biosciences, GE), 50 mM Tris–HCl, pH 8.0 using ÄKTA Purifier protein chromatography system (Amersham Biosciences, GE). The purity of the pooled fractions was checked by 15% SDS–PAGE, the concentration of proteins was determined using Kjeldahl
determination. MALDI-TOF-MS and Circular Dichroism (CD) were measured by Beijing Proteome Research center. Haemagglutination assay Haemagglutination activity was measured as described previously [2]. Fresh blood was obtained from Wistar rats and sheep stored in a specific pyrogen-free room. After washing 6 ml of whole blood with physiological saline solution three times, blood cells were collected by centrifugation at 500g for 10 min and then suspended to 1.0% (v/v) with physiological saline solution. Suspensions of 100 lL of purified reFIP-vvo (final concentration from 0.1 lg/mL to 5 lg/mL) in physiological saline solution, and 100 lL of 1.0% red blood cells placed in 96-well microtiter plates (round bottom, Nunc)
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were shaken for 30 s at room temperature and then incubated at 37 °C. The plates were examined for haemagglutination after 2 h. Mouse splenocytes proliferation assay Female BALB/c mice aged 8–12 weeks were maintained under standard pathogen-free conditions. Murine splenocytes were prepared as previously described [14] with minor modifications. Mouse spleens were surgically removed and minced at room temperature in RPMI 1640 medium. The tissue debris was removed by passing the cell suspension through a cotton column. Splenocytes (2 105 per well) were seeded in a 96-well plate, with a volume of 100 lL. Then 100 lL purified reFIP-vvo diluted in medium were added at the final concentrations of 0.3–5 lg/mL, incubated for 24 h. Finally 20 ll WST-1 was added to each well and incubated at 37 °C for another 2.5 h, and then the mouse splenocytes proliferation were quantified by measuring the absorbance at 450 nm. ConA (5 lg/mL) and medium with 1% PBS were applied as positive and negative controls, respectively. Results and discussion Expression of reFIP-vvo in X-33 The coding gene for reFIP-vvo was designed according to the codon usage of P. pastoris and was amplified by PCR using primers mentioned above from a synthesized vector bearing the DNA sequence of reFIP-vvo. To create the expression construct of pPICZaA-FIP-vvo (Fig. 1A), amplified gene of reFIP-vvo was cloned into the pPICZaA expression vector via the 50 EcoRI and 30 KpnI restriction sites. The pPICZaA bearing the DNA sequence of reFIP-vvo was linearized and transformed in the P. pastoris X-33 strain by electroporation. In the pPICZaA-FIP-vvo, the FIP-vvo gene was under the control of the AOX1 promoter allowed for the protein secretion in P. pastoris by induction with methanol and using the yeast signal a-factor included in the vector for extracellular targeting of the expressed protein. P. pastoris was cultivated on nutrient rich (BMGY/BMMY) media recommended by the supplier (Invitrogen, USA) for three days. Genomic DNA was isolated from clones to determine if the gene of interest has integrated into the Pichia genome. We amplify the gene of interest by PCR and analyzed on a 1% agarose gel (Fig. 1B). The supernatant of the clones with positive result in the PCR analysis were tested by western blot analysis (Fig. 1C). reFIP-vvo was expressed in culture media used and the yield of reFIP-vvo as shown in Fig. 2A increased upon induction by methanol at time point of 18, 24, 30, 36 and 42 h. Moreover, to the best of our knowledge, this is the first successful heterologous expression and production of FIP-vvo in a Pichia expression system to be reported.
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even more favorable. reFIP-vvo was obtained in a high yield and amount (Table 1). The purity of the reFIP-vvo was confirmed by SDS–PAGE analysis, showing a single band of 12 kDa, which is the same as for the wild type FIP-vvo. Initial expression experiments of transformed P. pastoris cells showed that the band of reFIP-vvo appeared at approx. 12 kDa in SDS polyacrylamide gel. The results of MALDI-TOF-MS confirmed the molecular weight of reFIP-vvo was approximately 12,658.20 Da (Fig. 3B), which correspond with theoretical Mr.12667.23 Da (http://expasy.org/cgi-bin/pi_tool). As described in the Methods, reFIP-vvo was purified to homogeneity in only one step as seen in Table 1. An overall concentration of 3.84 mg/ mL was achieved. As shown in Fig. 3A, the percentage of secondary structures in reFIP-vvo are 16.8% (a-helix), 31.8% (b-sheet), 16.8% (a-helix), 19.2% (b-turn), 32.2% (Random). Bioactivity of reFIP-vvo In vitro, the reFIP-vvo was capable of hemagglutinating red blood cells of Wistar rats (at concentrations greater than 0.2 lg/ml) and sheep (at concentrations greater than 1.0 lg/ml), but no such activity was observed toward human red blood cells, which were similar to the native FIP-vvo (Fig. 4). reFIP-vvo also plays a role as a mitogen in mouse spleen cells. The reFIP-vvo enhanced the proliferation of mouse spleen cells (Fig. 5). The mouse splenocyte proliferation activity of reFIP-vvo expressed in P. pastoris was much higher than the reFIP-vvo expressed in E. coli [15]. Now that the FIP-vvo gene has been expressed in the P. pastoris system, but more work needs to be done in improving the expression level and scaling-up to industrial production. The anti-tumor and anti-allergic activities in vivo also need to be further demonstrated. Additionally, the mechanism of how the FIP-vvo exert its effects on immune cells needs to be investigated and elucidated. Nevertheless, this study might supply some basis for the largescale production of recombinant FIP-vvo and its potential application in oral administration or therapeutic utilization in medical researches. Conclusions Here FIP-vvo gene from V. volvacea has been integrated into the yeast genome and expressed effectively at a high level (about 500 mg/L). The reFIP-vvo possessed very similar biological activities to native FIP-vvo, suggesting its potential application as a food supplement or immunomodulating agent in pharmaceuticals and even medical studies. References
Purification of the reFIP-vvo reFIP-vvo was purified from the culture medium of X-33 after three days of cultivation as described above. After filtration with a 0.22 lm filter, approximately 400 mL supernatant was dialyzed against water for 2 days and was loaded to the Hiload 26/20 SP Sepharose XL column. And purified further by Hiload 16/60 Superdex 75 column (Amersham Biosciences). The purity of the pooled fractions was checked by SDS–PAGE (Fig. 2B). The protein sample was concentrated to 3.8 mg/mL in the buffer of 50 mM NaCl, 20 mM Tris–HCl, pH 7.0 for further use. The isoelectric point was experimentally determined to be 7.83. Extracellular reFIP-vvo secreted by P. pastoris into medium was purified to homogeneity by a single cation exchange chromatography step, thus making the recombinant production of the protein
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