Appl Biochem Biotechnol (2014) 174:259–269 DOI 10.1007/s12010-014-1057-1

High-level Expression of a Manganese Superoxide Dismutase (PoMn-SOD) from Pleurotus ostreatus in Pichia pastoris Chaomin Yin & Wenxia Zhao & Liesheng Zheng & Liguo Chen & Qi Tan & Xiaodong Shang & Aimin Ma Received: 10 February 2014 / Accepted: 14 July 2014 / Published online: 25 July 2014 # Springer Science+Business Media New York 2014

Abstract The full-length cDNA of Pleurotus ostreatus superoxide dismutase (PoMn-SOD) was cloned and successfully expressed by using the pPIC9K vector under the control of alcohol oxidase 1 promoter with a secretion signal peptide (α-factor) in Pichia pastoris. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting demonstrated that recombinant PoMn-SOD, a 21.8 kDa protein, was secreted into the culture medium. Nondenaturing PAGE experiments confirmed that recombinant PoMn-SOD was secreted in a functionally active form and the expression system did not require any acid activation process. The factors affecting the expression level were optimized in shaking flask cultures. The maximum enzyme activity (156.9 U/mg) was observed under the following conditions: Initial medium pH was 6.0, induction time point was at the 6th day, and methanol concentration was 0.7 % (v/v). This was the first report on secretory expression of recombinant PoMn-SOD in P. pastoris, which might provide a reference for further practical applications. Keywords Pleurotus ostreatus . Manganese superoxide dismutase (PoMn-SOD) . Pichia pastoris . Expression . Optimized culture parameters . Polyacrylamide gel electrophoresis (PAGE)

Introduction Superoxide dismutase (SOD) is an abundant enzyme present in most aerobic and many anaerobic organisms [1]. The best-known activity of SOD is to catalyze the conversion of C. Yin : W. Zhao : A. Ma (*) Key Laboratory of Agro-Microbial Resources and Utilization, Ministry of Agriculture, College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China e-mail: [email protected] L. Zheng : L. Chen College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, China Q. Tan : X. Shang (*) National Research Center for Edible Fungi Biotechnology and Engineering, Key Laboratory of Applied Mycological Resources and Utilization, Ministry of Agriculture, Shanghai Key Laboratory of Agricultural Genetics and Breeding, Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, Shanghai 201403, China e-mail: [email protected]

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superoxide radical (O2−) to oxygen (O2) and hydrogen peroxide (H2O2) [2]. Therefore, SOD plays a crucial role in protecting biological systems against the damage mediated by this deleterious radical [3–6]. According to the metal embedded at the active site of SOD, it could be divided into copper- and zinc-containing SOD (Cu/Zn-SOD), iron-containing SOD (FeSOD), and manganese-containing SOD (Mn-SOD) [7]. Mn-SOD exists in the mitochondria of eukaryotic cells and the cytoplasm of many bacteria [2], and it has been considered to be a ubiquitous metallo-enzyme which is essential for the survival of all aerobic organisms [8]. The important function of Mn-SOD has led investigators to clone various Mn-SOD genes from animals [7, 9, 10], plants [11–13], and microorganisms [14–16]. In recent years, the expression of Mn-SOD by genetic engineering techniques has been investigated extensively. A variety of Mn-SODs from different species have been expressed in Escherichia coli, including Mn-SODs from Phascolosoma esculenta [7], Nelumbo nucifera [11], Mycobacterium sp. [14], Lactobacillus casei [15], Bacillus sp. [16], Danio rerio [17], Pistacia vera [18], Haliotis discus discus [19], Laternula elliptica [20], and Beauveria bassiana [21]. Additionally, Mn-SODs from Thermoacus aurantiacus [22] and Geobacillus sp. [23] have been expressed in the methylotrophic yeast Pichia pastoris, which is an excellent expression system with the advantages of posttranslational modifications and fast growth on economic salt-based media [24]. Pleurotus ostreatus, known as oyster mushroom, is the second largest commercially cultivated edible fungus in the world [25]. Up to now, a variety of compounds including lectins, polysaccharides, and polysaccharide-protein complexes from P. ostreatus have been reported to show the antioxidant effects [26]. However, the SOD of P. ostreatus, as an important antioxidant enzyme, has not been well documented. Previously, we have cloned and identified a manganese SOD gene (PoMn-SOD) (GenBank Accession No. KF768153) from P. ostreatus. In this paper, high-level expression of PoMn-SOD in P. pastoris has been described.

Materials and Methods Strains, Plasmids, and Reagents P. ostreatus Pd739 and E. coli DH5α were provided by Laboratory of Food Microbiology, Huazhong Agricultural University, and maintained on potato dextrose agar (PDA, Difco, USA) slant and Luria-Bertani (LB) medium (Difco, USA), respectively. P. pastoris GS115 was purchased from Invitrogen (Shanghai, China) and cultured in yeast extract peptone dextrose (YPD) medium (Difco, USA). Plasmids pMD®18-T (Takara, Dalian, China) and pPIC9K (Invitrogen, USA) were used as bacterial cloning vector and gene expression vector in P. pastoris, respectively. All restriction enzymes, Ex Taq™ DNA polymerase, DNA marker, and protein marker were purchased from TaKaRa (Dalian, China). Isolation of Total RNA and Amplification of PoMn-SOD Total RNA was extracted from P. ostreatus Pd739 mycelium using the TRIzol Reagent (Invitrogen, USA) according to the manufacturer’s protocol. Complementary DNA (cDNA) was synthesized using the PrimeScript™ Double-Strand cDNA Synthesis Kit (Takara, Dalian, China) according to the user manual. The PoMn-SOD cDNA was amplified using the specific primers PS-EF and PS-ER that contain the restriction enzyme sites (Table 1). The thermal cycling conditions were as follows: 94 °C for 10 min followed by 35 cycles at 94 °C for 30 s,

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58 °C for 30 s, and 72 °C for 1 min, with a final extension at 72 °C for 10 min. The PCR product was purified with AxyPrep™ DNA gel extraction kit (Axygene, Hangzhou, China), then cloned into pMD®18-T to form intermediate vector pMD-SOD, and transformed into E. coli DH5α for sequencing (Invitrogen, Shanghai, China). Construction of Expression Vector The plasmid DNA of pMD-SOD was extracted using the AxyPrep™ Plasmid Miniprep Kit (Axygene, Hangzhou, China) and digested with EcoRI and NotI, respectively. The target fragment was purified with AxyPrep™ PCR Cleanup Kit (Axygene, Hangzhou, China) and ligated into pPIC9K vector digested with the same two enzymes. The resulting plasmid pPIC9K-SOD contained the PoMn-SOD cDNA under the control of alcohol oxidase 1 promoter and was in frame of the α-factor secretion signal sequence (Fig. 1). After transformation, the positive colonies were checked for accurate insertion by sequencing. Transformation of P. pastoris Cells and Selection of High-Level Expression Colonies After linearized with PmeI and dephosphorylated with alkaline phosphatase (E. coli C75), the recombinant plasmid pPIC9K-SOD and the blank plasmid pPIC9K were electro-transformed into P. pastoris GS115 competent cells according to the EasyComp™ (Invitrogen, USA) procedure. After electroporation, 1 M ice-cold sorbitol was added immediately, and the cuvette contents were incubated at 28 °C for 60 min. The mixture was spread on YPD plate containing zeocin (0.1 g/L) and cultured at 28 °C for 2 days. After the transformants with zeocin resistance appeared, single colonies of transformants were picked up randomly and spotted on YPD plates containing geneticin 418 (G418, Sigma, USA) in different concentrations of 0.5, 0.75, 1.0, 2.0, and 3.0 mg/mL. The clones with the highest resistance to G418 were detected by PCR using primers α-factor and Aox1-3, combining with PoMn-SOD-specific primers PS-EF and PS-ER (Table 1) [27]. Expression of PoMn-SOD in P. pastoris GS115 For expression of PoMn-SOD, cells of His+ Mut+ transformants from a single colony of P. pastoris containing the pPIC9K-SOD vector were inoculated into 100-mL liquid BMGY medium [28], respectively. The cultures were grown at 28 °C in vigorous shaking (250 rpm) Table 1 Primers for PCR amplification in this study Primers Sequences (5′→3′) PS-EF PS-ER α-

Descriptions

Primers for PoMn-SOD cDNA, underlined parts indicate EcoRI and NotI restriction sites, respectively ATTTGCGGCCGCCTAATGATGATGATGA TGATGCGCAGAAGCTTCGAGGAAAC

CCGGAATTCATGAAGCACACCCTGCCT GAT

TACTATTGCCAGCATTGCTG

Primers for amplification of incorporation gene

factor Aox1-3 GCAAATGGCATTCTGACAT M13F

CGCCAGGGTTTTCCCAGTCACGAC

M13R

AGCGGATAACAATTTCACACAGGA

Primers for sequencing

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Fig. 1 Map of the pPIC9K-SOD expression vector

overnight. After the OD600 of cultures reached 2.0, the cells were collected (3,000 g, 5 min) and resuspended in 150-mL BMMY medium [28]. The cultures were maintained at 28 °C in a shaking incubator and supplemented with 100 % methanol daily to a final concentration of 0.5 %. One milliliter of the culture medium was sampled and centrifuged (8,000g, 20 min at 4 °C) each day. The supernatant was used for recombinant protein detection, and the strain containing blank plasmid pPIC9K was used as a negative control. SDS-PAGE and Western Blot Analysis Collected samples were analyzed by SDS-PAGE using 12 % polyacrylamide gels with a MiniProtean 3 Cell (Bio-Rad, USA). Samples were treated with 4× protein SDS-PAGE loading buffer (Takara, Dalian, China) in boiling water for 10 min before loading. The gel was run with 1× Tris-Glycine SDS running buffer for 2 h in 115 V. Then, it was washed and stained with Coomassie Brilliant Blue R-250 (Sigma, USA). To confirm that the recombinant SOD was a His-tagged fusion protein, Western blot analysis was performed using the mouse anti-His-Tag antibody (Tiangen, China) as a primary antibody and goat anti-mouse IgG conjugated with horseradish peroxidase (HRP) (Tiangen, China) as the secondary antibody. The specific operations for Western blot were described previously [28]. Verification of Recombinant PoMn-SOD Activity According to the modified procedure [29], nondenaturing polyacrylamide gel electrophoresis was carried out with 1.5 mm of 12 % polyacrylamide mini-slab gel in standard tris-glycine buffer (pH 8.3) at room temperature. Samples of 20 μL were loaded into each well and then electrophoresed at 60 V through the 5 % stacking gel for 45 min and 120 V through the separating gel for 2 h. After electrophoresis, a modified photochemical method was used to locate SOD activities on gel based on Beauchamp [30] and Elstner [31]. The gel was soaked first in 25 mL of 2.45 mM nitrotetrazolium blue chloride (NBT) for 45 min, washed briefly, then soaked in the dark in 30 mL of 50-mM potassium phosphate buffer (pH 7.8) containing 28 mM TEMED and 0.028 mM riboflavin for another 30 min. The gel was illuminated on a light box with an intensity of 30 μmol photons/m2/s1 until the activity bands were observed as

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pale zones on the dark blue background. The gels were photographed, and then kept in 50-mM potassium phosphate buffer (pH 7.8) in complete darkness. Activity Assay of Recombinant PoMn-SOD To detect the recombinant PoMn-SOD activity of cell-free extracts, Cu/Zn-SOD and Mn-SOD Assay Kit with WST-1 (Beyotime Institute of Biotechnology, Jiangsu, China) was applied. According to the protocol described in the kit, the absorbance was measured with a Thermo Multiskan Mk3 (Thermo Fisher Scientific, USA) at 450 nm in 96-well microtiter plates. One unit (U) of SOD activity was defined as the amount of enzyme that gave 50 % inhibition of NBT reduction. Protein concentration was determined by the Bradford method with BSA as a standard [32]. Optimization of Recombinant PoMn-SOD Expression in P. pastoris To achieve high-level expression of recombinant PoMn-SOD, different culture parameters including pH value, induction time, and methanol concentration were evaluated in the expression procedure. The initial medium pH value was set from 3.5 to 7.0 with an interval of 0.5 units by 0.1 M citric acid or 0.1 M sodium bicarbonate solution. After being induced by 0.5 % methanol, the samples were collected every 24 h. And, the samples which induced by different methanol concentrations, 0.5, 0.7, 1.0, 1.2, and 1.5 %, were collected until 144 h. The OD600 of the culture fluid and PoMn-SOD activity was monitored throughout the culture period.

Results Identification of Recombinant Plasmid and Recombinants The PoMn-SOD cDNA fragment of P. ostreatus had been amplified with the specific primers (Table 1). It contained 588 bp nucleotides encoding a protein of 195 amino acids with a predicted molecular weight of 21.8 kDa. After digestion with restriction enzymes, the fragment was ligated into pPIC9K vector. The recombinant plasmid pPIC9K-SOD was demonstrated to be correct by sequencing. After pPIC9K-SOD was electro-transformed into P. pastoris GS115 competent cells, dozens of transformants with zeocin resistance were obtained. PCR analysis showed that PoMn-SOD cDNA was indeed integrated into the genome of P. pastoris GS115 transformed with pPIC9K-SOD, while no band was found in the control sample, which was transformed with pPIC9K blank plasmid (data not shown). Expression of PoMn-SOD in P. pastoris After induction with methanol, five transformants showed high expression levels of PoMnSOD, and one of them was used for further experiments. Compared to the empty vector control, a band of 21 kDa from the supernatant of GS115 (pPIC9K-SOD) was detected on SDS-PAGE (lanes 1 and 3 in Fig. 2a). The estimated molecular weight of PoMn-SOD was consistent with the result of SDS-PAGE. With the extension of induction time, more and more proteins are expressed (lanes 4 and 5 in Fig. 2a). These results indicated that the SOD protein could be expressed normally in P. pastoris with the induction of methanol. The Western

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blotting result (Fig. 2b) further confirmed that the unique band was indeed the His-tagged fusion protein. Determination of Recombinant PoMn-SOD Activity We conducted the nondenaturing PAGE to detect the crude recombinant PoMn-SOD functional activity. In Fig. 3, the empty vector control showed no band while a clear band was observed from the supernatant of GS115 (pPIC9K-SOD). This result proved that the expressed product was indeed Mn-SOD and the recombinant PoMn-SOD was secreted in a functionally active form. Optimal Conditions of Recombinant PoMn-SOD Expression To overproduce the recombinant protein, the pH value, the induction time, and final methanol concentration of selected positive strain GS115 (pPIC9K-SOD) were optimized. After a series of experiments, the optimal expression conditions for recombinant PoMn-SOD were obtained as follows: The pH value was 6.0 (Fig. 4), the induction time was about the 6th day (Fig. 5), and methanol daily addition concentration was 0.7 % (v/v) (Fig. 6). Under these conditions, high-level expression transformant of P. pastoris strain was obtained, and the maximum activity of recombinant PoMn-SOD was 156.9 U/mg.

Discussion SOD enzymes play critical roles in defending against ROS and superoxide anion radicals, which generate mostly in mitochondria and result in cell dysfunction and death [6, 7]. MnSOD has the ability to catalyze the toxic superoxide anion into molecular oxygen and hydrogen peroxide; then, hydrogen peroxide will be removed by catalase and glutathione peroxidase [33]. Therefore, Mn-SOD can control the redox state to protect cells against the damage caused by ROS.

Fig. 2 SDS-PAGE and Western blot analysis of the recombinant PoMn-SOD after induction for 4, 5, and 6 days. a The SDS-PAGE analysis of the recombinant PoMn-SOD. M marker proteins, 1 supernatant from blank control, 2 supernatant from P. pastoris strain GS115 (pPIC9K-SOD) with no induction, 3–5 supernatant from P. pastoris strain GS115 (pPIC9K-SOD) after induced 4, 5, and 6 days. b Western blot analysis of the recombinant PoMnSOD. M marker proteins, 1 supernatant from blank control, 2 supernatant from induced P. pastoris strain GS115 (pPIC9K-SOD). The marker proteins comprised phosphorylase B (97 kDa), albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (29 kDa), and trypsin inhibitor (20 kDa)

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Fig. 3 Detection of the recombinant PoMn-SOD activity by polyacrylamide gel electrophoresis (PAGE) after induction for 3 and 6 days. 1 Supernatant from blank control, 2 supernatant from GS115 (pPIC9k-SOD) after induction of 3 days, 3 supernatant from GS115 (pPIC9k-SOD) after induction of 6 days

Mn-SOD from P. ostreatus has seldom been studied to our knowledge. In this study, we expressed P. ostreatus Mn-SOD (PoMn-SOD) in P. pastoris system. SDS-PAGE analysis showed that the recombinant PoMn-SOD protein was approximately 21 kDa; this was similar to the molecular mass of Mn-SOD from Bacillus sp. MHS47 [16], Thermoascus aurantiacus [22], and Chaetomium thermophilum [34]. Western blot analysis showed that the anti-His-Tag antibody generated in mouse reacted with the recombinant PoMn-SOD and created specific signals. This result demonstrated that the recombinant PoMn-SOD was indeed the His-tagged fusion protein with no frame shift problem occurring in translation and it had been successfully expressed in P. pastoris. Secretory expression of the target protein usually helps subsequent purification process [35]. The target protein may degrade during secretory expression especially in high-cell density culture, which often hinders effective purification of target protein [36, 37]. In our study, the clear target band was observed in SDS-PAGE, and unique band appeared in Western blot analysis (Fig. 2). This showed that the recombinant PoMn-SOD did not degrade. As a

Fig. 4 The optimal pH value for the recombinant PoMn-SOD expressed in P. pastoris. The samples were collected after 24 h induced by 0.5 % methanol

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Fig. 5 The optimal induction time for the recombinant PoMn-SOD expressed in P. pastoris. The samples were collected after every 24 h induced by 0.5 % methanol with pH value of 6.0

eukaryotic expression system, P. pastoris was powerful in posttranslational modifications that included polypeptide folding, glycosylation, methylation, acylation, proteolytic adjustment, and targeting to subcellular compartments [38]. The result of nondenaturing PAGE experiments confirmed that recombinant PoMn-SOD was secreted in a functionally active form

Fig. 6 The optimal methanol concentration for the recombinant PoMn-SOD expressed in P. pastoris. The samples were collected after 144 h with pH value of 6.0

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which did not require any downstream manipulations, such as solubilization and refolding [39]. Several factors affect production yield during the expression of recombinant protein in P. pastoris, including culture pH, induction time, final methanol concentration, and so on. The kinetics of proteolytic reactions, in the presence or absence of cells, is shown to be influenced by pH [38]. Optimum pH is critical for cell growth, protein expression, and protein stability [40]. Although P. pastoris is capable of growing across a relatively broad pH range (3.0–7.0), different pH values are found to be optimal according to the recombinant protein’s stability [37]. pH 3.0 is optimal for production of aspartic proteinase from Mucor mucedo [40], while pH 5.0 is optimal for production of recombinant Cu/Zn-SOD from Cervus nippon [41]. It has been reported that the optimum pH for Mn-SOD activity in C. thermophilum [34] and T. aurantiacus [22] was 7.5. However, studies conducted by Ken et al. [42] show that acidic pH rather than nonacidic pH was favorable to form monomer of the protein. In our study, maximum activity of the recombinant PoMn-SOD was observed when pH value is 6.0. Besides, induction time is another factor that influences the yield of recombinant protein. In P. pastoris expression system, the maximum production of recombinant Cu/Zn-SOD from Saccharomyces cerevisiae appears to be at 72 h after induction [1], but maximum activity and dry cell weight of Cu/Zn-SOD from C. nippon were achieved after induction for 168 h [41]. Our data shows that the SOD activity and OD600 of the recombinant PoMn-SOD increase continually until induction for 144 h. Then, the SOD activity will decrease dramatically while OD600 keeps on increasing (Figs. 5 and 7). So, we consider that induction for 144 h is optimal for the recombinant PoMn-SOD. A methanol induction strategy is critical for successful expression of heterologous proteins in P. pastoris [38], and high methanol concentration has a harmful influence on the cells [27]. After analysis of SOD activity and OD600 data, we chose 0.7 % methanol as the suitable concentration for PoMn-SOD production.

Conclusion For the first time, we successfully expressed PoMn-SOD gene from P. ostreatus in P. pastoris. The recombinant PoMn-SOD was secreted in a functionally active form with the maximum crude activity of 156.9 U/mg. Additionally, the culture conditions had been further optimized which would provide a good basic support for further investigations on the large-scale

Fig. 7 SDS-PAGE analysis of the recombinant PoMn-SOD expressed in P. pastoris in the optimum conditions. M marker proteins, 1–6 supernatants from P. pastoris strain GS115 (pPIC9K-SOD) after induction of 1–6 days

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fermentation experiments and other practical applications. Currently, we are trying to purify the recombinant PoMn-SOD with the 6× His affinity tag, and the enzymatic properties will be further investigated. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (31172011) and the Chinese National Science and Technology Support Program (2013BAD16B02). We thank Qin Wang and Jihong Zhu for assistance in conducting Western blot experiment and reviewing the manuscript.

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High-level expression of a manganese superoxide dismutase (PoMn-SOD) from Pleurotus ostreatus in Pichia pastoris.

The full-length cDNA of Pleurotus ostreatus superoxide dismutase (PoMn-SOD) was cloned and successfully expressed by using the pPIC9K vector under the...
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