Western com rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is the most economically important insect pest of com plants, Zea mays (L.) in the United States (Levine and Oloumi-Sadeghi 1991). Recently, it was shown that not only endogenous endoglucanase activity is present in three gut regions of this insect pest but also Dw-ENGase I, a ß-l,4-endoglucanase (ENGase), is a monomeric protein with a molecular mass of 26 kDa that belongs to the glycoside hydrolase family 45 (GHF45), a large and growing hydrolase family (Valencia Jiménez et al. 2013). It has been shown that the presence of one or more possible posttranslational modifications may be necessary for secretion and biological activity of recombinant cellulases that have been cloned and expressed from other coleopteran insects (Wei et al. 2005, 2006). The Dvv-ENGase I identified from D. v. virgifera contains a possible posttranslational modification at the N-glycosylation site, 100–102 (N-S-T) in the amino acid sequence (Valencia Jiménez et al. 2013), which could affect the final biological activity of the recombinant enzyme, especially if a prokaryotic expression system is used.

For many years, Escherichia coli-based expression systems have been the most common method for the expression of recombinant proteins due to rapid cell growth, inexpensive cell culture media, and relative ease of genetic transformation (Sivashanmugam et al. 2009, Makino et al. 2011). However, bacterial-based systems can generate misfolded proteins as well as the formation of inclusion bodies and thus may yield nonfunctional proteins (Biely et al. 1985). In addition, some reports have described recombinant proteins that do not fold properly in bacterial systems but which can be expressed successfully in yeast systems (Cereghino and Cregg 2000, Wysocka-Kapcinska et al. 2010). In recent years, the methylotrophic yeast, Pichia pastoris, has become a successful and useful biotechnological tool for the expression of different heterologous proteins (Cregg et al. 1987, 1993). P. pastoris provides a eukaryotic expression system that is not only well known as an efficient system for expression and secretion of heterologous proteins (Cereghino and Cregg 2000) but also as an alternative to E. coli for expression of eukaryotic proteins that may require posttranslational modification (Wysocka-Kapcinska et al. 2010). P. pastoris has been successfully used to express proteins for both basic research and industrial uses (Higgins and Cregg 1998), using low-cost and well-established culture methods to produce large amounts of a recombinant product based on the presence of a highly expressed alcohol oxidase promoter (Cregg et al. 1987, 1993; Canales et al. 1997; Stratton et al. 1998). A number of different recombinant proteins from a variety of eukaryotic sources have been successfully produced in this eukaryotic expression system (Cereghino and Cregg 2000).

P. pastoris provides an important expression system or a variety of reasons including: 1) it is easily genetically manipulated, 2) it is capable of producing recombinant proteins in higher yields, and 3) it is capable of structural and posttranslational protein modification including glycosylation, disulfide-bond formation, and proteolytic processing and protein folding (Cregg et al. 2000). As a consequence, some proteins that cannot be expressed efficiently in other expression systems may be successfully produced as functional recombinant proteins in P. pastoris (Cereghino et al. 2002). P. pastoris is generally considered to be a recombinant culture system that is capable of producing proteins in higher yields than mammalian or insect cell expression systems (Higgins and Cregg 1998). Here we present the expression and biological characterization of an active form of Dvv-ENGase I using the eukaryotic expression system of P. pastoris GS 115 and demonstrate the utility of P. pastoris as an appropriate system for expression of insect heterologous proteins.

Materials and Methods

Strains and Reagents. pEXP5-CT/TOPO-DvvENGI, a plasmid containing the gene for expression of an endogenous ENGase (DvvENGase I), was obtained from an E. coli colony that was previously transformed in our laboratory and kept at -80°C. P. pastoris GS115, the Pichia EasyComp Kit, and pPICZαA vector were purchased from Invitrogen (Carlsbad, CA). Restriction enzymes were purchased from Fermentas (Hanover, MD). Carboxymethyl cellulose (CMC) and ostazin brilliant red-hydroxyethyl cellulose (OBR-HEC) were purchased from Sigma Chemical (St. Louis, MO).

Construction and Cloning of the Recombinant Expression Plasmid. The coding region of Dvv-ENGase I gene was amplified by polymerase chain reaction (PCR) from plasmid pEXP5-CT/TOPO-DvvENGI using the following two primers: pEXP5 forward [5-GAATTCATGAAGATCGCTATCCTT-3] (EcoRI) and pEXP5 reverse [5-GCGGCCGCTTAAAGACTGCAACCA-3] (NotI) (restriction sites are underlined and the reverse stop codon TTA is bold). The PCR product containing the Dvv-ENGase I was submitted to double digestion with EcoRI and NotI restriction enzymes and ligated into pPICZαA vector which utilizes the AOX1 promoter of P. pastoris and the alphafactor leader sequence from Saccharomyces cerevisiae for final product secretion. Finally, the plasmid pPICZαA-DvvENGI (20 µg) was linearized using SacI, and the DNA product was used to transform P. pastoris GS115 competent cells.

Preparation and Transformation of Competent Pichia Cells. The preparation and transformation of competent Pichia cells was done using the Pichia EasyCompKit (Invitrogen Carlsbad, CA). In general, transformation was performed by using 50 µl of competent cells with 3 µg of linearized plasmid DNA. Transformation efficiencies may vary not only based on the strain that is used but also on the efficiency of plasmid integration into the Pichia genome. The integration of the linearized pPICZαA-DvvENGI plasmid into the Pichia genome was confirmed by cloning PCR according to the manufacturer's instructions using the 5′ AOX1 primer paired with the 3′ AOX1 primer included in the kit. Recombinant DNA manipulations were carried out in E. coli TOP 10 chemical competent cells (Invitrogen). E. coli cells were cultured in LB medium (0.5% yeast extract, 1% glucose, and 0.5% NaCl) at 37°C with antibiotics at the following final concentrations: 100 µg/ ml ampicillin and 25 µg/ml Zeocin for plasmid selection. Plasmid DNA was purified from E. coli cell cultures using a QIAprep Spin Miniprep Kit (Qiagen, Chatsworth, CA). DNA digested with restriction enzymes was resolved on TBE (Tris-Borate-EDTA buffer) agarose gels and purified using the QIAquick Gel extraction kit (Qiagen, Chatsworth, CA). A P. pastoris colony that was transformed with the empty pPICZαA vector was used as a control for documenting expression of a functional protein.

Small Scale Expression. Transformants were selected on YPD plates (1% yeast extract, 2% peptone, and 2% glucose) containing 100 µg/ml Zeocin as the selective marker. Positive P. pastoris transformants were grown in BMGY medium (buffered glycerol-complex medium, containing 1% glycerol, 2% peptone, and 1% yeast extract) at 30°C and 250 rpm until an OD600 of approximately 2–6 was reached ( ∼16–18 h). The Pichia cells were harvested by centrifugation at 2,500 × g for 5 min at room temperature (RT), and the supernatant was decanted and the cell pellet resuspended to an OD600 of 1.0 in BMGY medium to induce expression (approximately 100–200 ml). Induction was initiated by adding 100% methanol to a final concentration of 0.5% methanol every 24 h. At 24-h intervals after initiating induction, 1 ml aliquots of the expression culture samples were collected in a 1.5-ml microcentrifuge tube and centrifuged at 15,000 × g at RT for 2–3 min. Supernatants were immediately frozen and stored at — 80°C and then used to analyze expression levels and determine the optimal time for post-induction harvest. A similar induction system containing protease inhibitors was tested in order to evaluate the effect of endogenous proteolytic activity on the enzymatic activity from the recombinant Dvv-ENGase I expressed using P. pastoris GS 115.

SDS-Polyacrylamide Gel Electrophoresis and Western Blotting. SDS—polyacrylamide gel electrophoresis (PAGE) was performed according to Laemmli (1970). All protein samples were diluted in 2 × SDS-PAGE sample buffer and separated on 10% SDS-PAGE. Proteins in the SDS-gel were electrophoretically transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA) using transfer buffer (25 mM Tris and 192 mM glycine in 20% methanol) at 90 mA overnight at 4°C. After blocking overnight at 4°C with 5% (w/v) nonfat dry milk prepared in TTBS (Tris-buffered saline, containing 150 mM NaCl, 20 mM Tris, 0.05% Tween-20, pH 7.5), the membranes were incubated for 1 h at RT with an anti-DvvENGase I polyclonal antiserum (1:10,000 v/v), generated using a 19 amino acid synthetic peptide (T P L E Q S P E I K F I E K G I S G E), which was designed based on the protein target sequence and using PeptideSelect Online (Invitrogen, Carlsbad, CA; Valencia Jiménez et al. 2013). After five washes with TTBS, the nitrocellulose membrane was incubated for 1 h at RT with anti-rabbit IgG antibody conjugated with alkaline phosphatase (1:10,000 v/v, Sigma-Aldrich Life Science). Finally, the membrane was washed three times and the specific protein bands were visualized by using Sigma Fast 5-bromo-4-chloro-3′-indolyphosphate/ Nitro-blue tetrazolium (BCIP/NBT; Sigma-Aldrich, St. Louis, MO). Precision Plus protein dual color standards (broad range, 10–250 kDa) were used as a control for protein transfer and estimation of molecular mass.

OBR-HEC Hydrolyzing Activity. ENGase was assayed in 50 mM sodium acetate (pH 5.0) with OBR-HEC (Sigma, St. Louis, MO) as substrate (Biely et al. 1985). The enzyme fraction (10 µl) was incubated with 50 µl of OBR-HEC solution (5 mg/ml) in a final volume of 250 µl for 15 min at 45°C. After 30 min, the reaction was stopped by adding 900 µl of ethanol. The reaction mixture was then centrifuged at 10,000 × g and the final absorbance was read at 550 nm. The enzyme activity was expressed as the AA550/min. One unit of OBR-HECase activity was defined as the amount of enzyme changing the absorbance (0.001/h).

Monitoring of Reducing Sugar Accumulation. The recombinant ENGase activity was also determined by measuring reducing sugar concentration using the dinitrosalicylic acid (DNS) method (Miller 1959). After 5 d of P. pastoris GS 115 culturing in a cell culture medium containing the substrates CMC and HEC (1%), 100 µl of supernatant was collected from each treatment, centrifuged at 10,000 × g for 15 min at RT and combined with 100 µl of 1% DNS (DNS containing 30% potassium tartrate and 0.4 M sodium hydroxide) and then boiled for 10 min at 100°C. Finally, the absorbance was determined at 550 nm. The recombinant ENGase activity was also determined using a modification of the method described in Mandels et al. (1976). Briefly, two filter paper discs (Whatman No. 1, 42.5 mm, GE Healthcare, Buckinghampshire, UK) were incubated with 10 ml of the 6-d P. pastoris cell culture supernatant in 40 ml of 50 mM citrate buffer pH 5.0 and incubated at 45°C for six consecutive days. Aliquots (100 µl) of the final incubation mix were collected daily, centrifuged at 10,000 × g for 15 min at RT and combined with 100 µl of 1% DNS. The final absorbance was determined at λ550 nm after boiling for 5 min at 100°C. The final ENGase activity was expressed as relative reducing sugar accumulation (%). Each experiment was carried out in triplicate.

ENGase Activity in Native Polyacrylamide Gels. Zymograms for ENGase activity were conducted under semidenaturing conditions on 7.5% SDS-PAGE gels containing 0.25% of copolymerized HEC (Fluka, Milwaukee, WI). After electrophoretic separation, the SDS gel was washed in 1% Triton X-100 solution for 30 min at 4°C followed by incubation in 50 mM sodium acetate buffer solution (pH 5.0) at 45°C to allow the enzyme to digest HEC. Finally, the gel was stained with 0.1% Congo red for 1 h at RT and destained in 1 M NaCl. The reaction was stopped by immersing the gel in a 10% (w/v) acetic acid solution and then photographed. A clear zone on a dark blue background indicated the hydrolysis of HEC as a result of ENGase activity.

Radial Diffusion Assay to Detect ENGase Activity. HEC-agar plates were prepared by dissolving agarose (1%), Congo red (0.02%), and HEC (0.5%) in 50 mM sodium acetate buffer (pH 5.0). After solidification into Petri plates to a depth of 10 mm, circular wells were punched into the agar with a 5-mm diameter cork borer. To detect the recombinant ENGase activity, 50 µl of P. pastoris cell culture supernatant was added to wells and incubated at 45°C overnight. After 24 h, the residual Congo red dye was removed by rinsing the plate with distilled water and then fixed by flooding the plate with dilute acetic acid (10%) for 1 h at RT. ENGase activity zones appeared as white haloes on a dark blue background.

Fig. 1.

Western-blot analysis of proteins from the supernatant of a P. pastoris cell-culture. P. pastoris GS115 was transformed with the pPICZαA vector containing the endoglucanase gene (DvvENGl) from D. v. virgifera. Lane 1, molecular mass markers; Lanes 2–7 represent fractions of cell culture fractions of 6 d of continuous cell-culture. Every 24 h the concentration of methanol was adjusted to 0.5% to maintain expression of the recombinant protein. A polyclonal antibody against an endoglucanase from D. v. virgifera was used as the primary antibody. The protein samples were run using a 10% SDS gel.

Protein Determination. The protein concentration was determined by the method of Bradford (1976) using bovine gamma globulin as protein standard. Protein samples were incubated for 15 min at RT and then read at 595 nm.

pH Optimum. The optimal pH was determined by preincubating 10 µl of the recombinant ENGase for 15 min at 45°C in appropriate buffers: 50 mM sodium phosphate (pH 2 and 7), 50 mM sodium citrate (pH 3, 4 and 6), 50 mM sodium acetate (pH 5), and 50 mM Tris-HCl (pH 8 and 9) and then starting the enzymatic reactions by adding 50 µl of OBR-HEC (5 mg/ml) as substrate. After 30 min, the reaction was stopped adding 900 µl of ethanol and then centrifuged at 10,000 × g at RT, and the final absorbance was read at 550 nm. Each pH determination was replicated three times.

Results

Expression of Dvv-ENGase I From P. pastoris. P. pastoris GS115 cells were transformed with the linearized pPICZαA-DvvENG I plasmid, which contained the coding sequence for DvvENG I gene from D. v. virgifera and then plated on YPD plates containing zeocin as a selective marker for positive colony selection. The expression of the recombinant protein was induced by adding methanol to the cell-culture every 24 h for 6 d and then the supernatant was collected daily for SDS-PAGE and western blot analyses. Western blotting analysis indicates that the secreted recombinant DvvENG I protein has an apparent molecular mass of 29 kDa and is clearly visible in the growth medium after 3 d of induced expression (Fig. 1). In addition, the accumulation of the recombinant protein during cell culture was also confirmed by enzymatic assays in which OBR-HEC was used as a substrate (Fig. 2). The accumulation of recombinant protein increased each day and reached a maximum by 6 d after induction.

Characterization and Biological Activity of the Recombinant Protein. The specific biological activity of the recombinant ENGase was evaluated by testing its capacity to digest the substrates including HEC, CMC, and Whatman No. 1 filter paper. Reducing sugar accumulation (%) was identified by incubating filter paper discs with the 6-d P. pastoris cell culture supernatant that contained the secreted recombinant enzyme (Fig. 3). The reducing sugar accumulation (%) was significantly increased when the cell culture medium was originally supplemented with CMC (1%) or with HEC (1%), although a higher accumulation of reducing sugar was evident when the expression medium contained HEC compared with CMC. No reducing sugar accumulation was observed for a nontransformed P. pastoris colony or a transformed P. pastoris colony carrying the empty vector grown in a medium supplemented with HEC (1%) (Fig. 4). The recombinant ENGase exhibited optimal enzymatic activity at pH 5.0 using OBR-HC as a substrate and appropriate buffer solutions (Fig. 5).

Fig. 2.

Enzymatic activity from Dvv-ENG I recombinant protein expressed using P. pastoris GS115 after 5 d of Cell-culture. The enzymatic reaction was initiated by adding 50 µl of OBR-HEC (5 mg/ ml) as substrate. One unit of OBR-HECase activity was defined as the amount of enzyme changing the absorbance (0.001/h).

Fig. 3.

Relative reducing sugar accumulation (%) during the degradation of filter paper (Whatman No. 1) by the enzymatic action of a recombinant endoglucanase gene from D. v. virgifera expressed in P. pastoris GS115. Two filter paper discs (Whatman No. 1; 42.5 mm) were incubated with 10 ml of 6-d P pastoris supernatant and 40 ml of 50 mM citrate buffer pH 5.0 and then incubated at 45°C for six consecutive days. The final absorbance was determined at λ550 nm after adding 500 µl of distilled water. Each experiment was carried out in triplicate.

ENGase Activity on HEC-Based Gels. A single enzymatic activity band was identified (Fig. 6A) after electrophoretic separation under semidenaturing conditions with SDS-PAGE gels containing 1% HEC (Fig. 6A; lanes T1–T4). This band was absent in the supernatant from the P. pastoris colony that was transformed with the empty pPICZαA vector (lanes E1–E4). The expressed protein also showed enzymatic activity based on the zone of clearing in the radial diffusion assays (Fig. 6B), and the activity was clearly enhanced by the presence of protease inhibitors based on the increased size of the clear zone (Fig. 6B1).

Discussion

We have previously reported the cloning and functional characterization of the Dvv-ENG I gene, which codes for an ENGase belonging to GHF45 from D. v. virgifera (Valencia Jiménez et al. 2013). It is important to note that high-level expression of recombinant ENGase’s from different insect sources has been efficiently achieved using either E. coli or the aforementioned P. pastoris expression system (Girard and Jouanin 1999, Lee et al. 2004, Inoue et al. 2005, Wei et al. 2005). The P. pastoris expression system is a widely reported method for the efficient production and secretion of recombinant proteins (Cregg et al. 2000) that can be expressed either extracellularly or intracellularly with high yields using well-established culture conditions. This recombinant expression system is also known for being a superior alternative to E. coli-based expression systems because of its natural capacity to express recombinant proteins that include posttranslational modifications not possible with E. coli-based expression (WysockaKapcinska et al. 2010). The production of a functionally active form of the recombinant ENGase Dvv-ENGase I using the P. pastoris GS115 strain for expression constitute the successful expression of this protein in a eukaryotic system.

Fig. 4.

Reducing sugar accumulation (%) during the culture of P. pastoris GS115 that was transformed with the Dvv-ENG I gene from D. v. virgifera. After 5 d of culturing, 100 µl of supernatant were collected from each treatment and combined with 100 µl of 1% DNS and then boiled for 5 min at 100°C. The final absorbance was determined at λ550 nm. HEC, P. pastoris cell culture containing 1% of HEC. CMC, P. pastoris cell culture containing 1% of CMC. EV, P. pastoris GS115 strain transformed with the empty vector as control. Each experiment was carried out in triplicate.

Fig. 5.

Effect of the pH on the enzymatic activity of recombinant endoglucanase from D. v. virgifera that was expressed in P. pastoris GS115 cell-culture after 6 d of cell-culture. The enzymatic reaction was initiated by adding 50 µl of OBR-HEC (5 mg/ml) as substrate. Each point is the average of three measurements.

The recombinant Dvv-ENG I protein was detected by SDS-PAGE and western blotting and indicates that the apparent molecular mass of the recombinant ENGase from P. pastoris (29 kDa) was greater than the predicted molecular mass of 26-kDa from the translated cDNA sequence (Valencia Jiménez et al. 2013). This difference in the molecular mass may be due to the posttranslational modifications, such as glycosylation, which can increase the apparent molecular mass in SDS-PAGE gels. It is well known that P. pastoris is able to add both O-linked and N-linked carbohydrate moieties to secreted proteins (Sivashanmugam et al. 2009).

Fig. 6.

(A) Endoglucanase activity assay on 7.5% semidenaturing SDS-PAGE gels containing 0.25 % (w/v) HEC of two P. pastoris cell cultures. Lanes E1–E4 represent four different protein concentrations from the supernatant of a P. pastoris GS115 strain that was transformed with the pPICZαA empty vector. Lanes T1–T4 represent four different protein concentrations from the supernatant of a P. pastoris GS115 strain that was transformed with the pPICZαA vector containing the endoglucanase gene (DvvENGl) from D. v. virgifera. The clear zones at the bottom of the gel indicate the hydrolysis of HEC as a result of endoglucanase activity. (B) Radial diffusion assay using agarose medium containing 0.5 % (w/v) HEC and 0.1% of Congo red. Samples were incubated at 45°C overnight; 1) 50 µl of total extract from P. pastoris cell culture containing protease inhibitors; 2) 50 µl of total extract from P. pastoris cell culture without protease inhibitors.

The enzymatic activity of the recombinant ENGase Dvv-ENG I from P. pastoris was higher than the biological activity of the recombinant ENGase that was initially expressed using the E. coli cell-free expression system (data not shown). This increased enzymatic activity from the recombinant ENGase expressed in P. pastoris may be related to a potential N-glycosylation site at the Dvv-ENGase protein structure. It has been shown that the enzymatic activity of recombinant insect cellulases, with the presence of one or more possible N-glycosylation sites, is essential not only for enzymatic activity but also for stability of the recombinant protein (Howard et al. 1991, Flesher et al. 1995, Wei et al. 2005, 2006).

The recombinant ENGase Dvv-ENG I was successfully expressed under the control of the alcohol oxidase (AOX1) promoter, which is strongly repressed in cells grown on glucose, but is highly expressed when cells are grown in a cell culture medium containing methanol as a main carbon source (Cereghino et al. 2002). Monitoring of reducing sugar accumulation was possible by adding supplemental HEC and CMC to the original P. pastoris cell culture medium. The reducing sugar accumulation (%) in the medium supplemented with HEC was significantly greater when compared with the P. pastoris cell culture medium that was prepared with CMC as an additional ingredient. This phenomenon can be explained by either a higher recombinant ENGase activity or an easier digestion of HEC, which is a nonionic substituted cellulose that has been recommended to determine ENGase activity (Wood and Bhat 1988). It is important to point out that no significant accumulation of reducing sugar was observed for transformed P. pastoris expressing the empty vector (data not shown).

By using appropriate buffer solutions and reaction conditions, it was found that the ENGase activity of the recombinant DvvENG I protein expressed in P. pastoris GS 115 cells has an optimum pH around 5.0. This result is similar to the previous report of enzymatic properties on optimum pH of the Ag-EGase I from Apriona germari (Lee et al. 2004) and from the mollusk Ampullaria crossean (Guo et al. 2008).

The higher enzymatic activity that was detected in the supernatant from the P. pastoris cell medium supplemented with protease inhibitors could be explained by susceptibility of the secreted recombinant ENGase to endogenous neutral proteolysis. Because part of the expressed protein is degraded during its expression, an unbuffered medium may then be important for future recombinant protein expression experiments. As Pichia expression progresses in an unbuffered medium, the pH becomes more acidic, inactivating many neutral pH proteases without affecting the cell growth (Brierley et al. 1994).

In conclusion, we have established an expression system using the methylotrophic yeast, P. pastoris GS115 strain, which will facilitate the production and expression of large amounts of a soluble and functionally active form of DvvENG I that will allow further biochemical and biophysical studies. Expression of insect ENGases and other cellulase genes should help develop an understanding about the role of insect cellulolytic enzymes during the cellulose hydrolysis that takes place in the insect gut.