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Enhancing chimeric hydrophobin II-vascular endothelial growth factor A165 expression in Pichia pastoris and its efficient purification using hydrophobin counterpart
International Journal of Biological Macromolecules 139 (2019) 1028–1034
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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac
Enhancing chimeric hydrophobin II-vascular endothelial growth factor A165 expression in Pichia pastoris and its efficient purification using hydrophobin counterpart Sareh Arjmand a,⁎, Zahra Tavasoli a, Seyed Omid Ranaei Siadat a,⁎, Behanm Saeidi a, Hossein Tavana b a b
Protein Research Center, Shahid Beheshti University, G. C., Tehran, Iran Department of Biomedical Engineering, The University of Akron, Akron, OH 44325, USA
a r t i c l e
i n f o
Article history: Received 14 May 2019 Received in revised form 3 August 2019 Accepted 8 August 2019 Available online 09 August 2019 Keywords: Vascular endothelial growth factor Hydrophobin Pichia pastoris Media optimization Protein purification
a b s t r a c t We report cloning and expressing of recombinant human VEGF-A165, fused at the N-terminal with Hydrophobin II (HFBII) from Trichoderma reseei, in yeast Pichia pastoris. We validated the construct using SDS-PAGE and ELISA against VEGF-A165 and efficiently performed protein purification and enrichment based on HFBII counterpart and using an aqueous two-phase system (ATPS) with nonionic surfactant X-114. We studied the effects of various culture medium additives and interaction effects of positive factors to increase the recombinant HFBII-VEGFA165 production. Supplementing the Pichia pastoris cell culture medium with Mg2+, Polysorbate 20 (PS 20), and 4-phenylbutyrate (PBA) improved the expression of the chimeric protein. Orthogonal experiments showed that the optimal condition to achieve maximal HFBII-VEGF-A165 production was with the addition of PBA, PS 20, and MgSO4. Under this condition, the production of the target protein was 4.5 times more than that in the medium without the additives. Overall, our approach to produce chimeric HFBII-VEGF-A165 and selectively capture it in ATPS is promising for large-scale protein production without laborious downstream processing. © 2019 Elsevier B.V. All rights reserved.
1. Introduction VEGF-A is a dimeric, disulfide-linked glycoprotein with key functions in both physiologic and pathologic neovascularization and angiogenesis. VEGF-A triggers multiple downstream signaling pathways by conjugating with the endothelial cell surface receptors including VEGF receptors 1 and 2 (VEGFR1 and VEGFR2) and the co-receptor neuropilin 1. VEGF-A has widely-expressed and tissue-specific isoforms, which are generated by alternative splicing and have different binding affinities to the VEGF receptors [1]. VEGF-A165 is the most abundant isoform that has binding affinity to all VEGF receptors. It plays a pivotal role in vascular development during embryonic development and wound healing [2]. This isoform also binds to heparin and heparin sulfate and regulates multiple aspects of angiogenesis and vasculogenesis [3]. VEGF-A165 is involved in cancer progression and several VEGF-A165 inhibitors have been approved for clinical use [4,5]. Additionally, this protein is useful for clinical applications such as wound healing, bone formation during bone repair, and prevention and treatment of ischemic heart disease [6–8]. Recombinant production and purification of VEGF-A165 and ⁎ Corresponding authors at: Protein Research Center, Shahid Beheshti University, G. C., PO Box 1983969411, Tehran, Iran. E-mail addresses: [email protected] (S. Arjmand), [email protected] (S.O. Ranaei Siadat).
https://doi.org/10.1016/j.ijbiomac.2019.08.080 0141-8130/© 2019 Elsevier B.V. All rights reserved.
related isoforms in different eukaryotic and prokaryotic expression systems with yields from micrograms to grams have been reported [9–12]. However, there is still a need for studies to optimize different steps of protein expression and purification to maximize the production efficiency and develop an economic and efficient industrial-scale production process. Optimization of media composition and/or addition of chemical supplements to growth media are among the traditional strategies to address this need. Numerous chemical additives have been shown to increase recombinant protein stability, solubility, folding, and expression. Among them, non-detergent surfactants, chemical chaperones, metal ions, and organic compounds have been widely studied. Positive effects of chemical additives are usually observed in certain ranges depending on the method of choice [13–16]. Optimization of the purification process is also used to increase the yield of recombinant protein production. Using purification tags as the fusion partner greatly simplifies the purification, although potential effects on the structure and biological activity of the target protein and stimulating immunogenic responses are the major drawbacks. For therapeutic proteins, it is usually necessary to remove the tag, but this results in extra steps and greater expense of production [17]. In this study, we considered the hydrophobin as a non-immunogenic, coexpressed fusion partner of VEGF-A165 for protein purification. Hydrophobins are a family of small, surface active, self-assembling
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proteins, which are produced by filamentous fungi and play a crucial role during the fungal life cycle. Hydrophobins share a unique structure consisting of four intramolecular disulfide bridges and exposed hydrophobic patches that are responsible for their amphipathic property [18–20]. These unique properties make hydrophobins desirable for many bio-industrial applications [21–24]. Hydrophobins are divided into two classes based on solubility and elasticity of their films: class I such as EAS from Neurospora crassa, and class II such as HFBI and HBFII from Trichoderma ressei [25]. This study focuses on HFBII due to its high aqueous solubility and film elasticity. Hydrophobins have been used as a fusing self-assembled tag in the expression and purification of several recombinant proteins, usually in plants, with considerable advantages such as high selectivity, high yield, and ease of use in the purification process [26–28]. Hydrophobin fusion technology has relied mainly on HFBI. It was recently shown that HFBII has properties such as anti-oxidant activity and Angiotensin Converting Enzyme (ACE) inhibitory effects [29], making it a suitable candidate as a fusion partner for therapeutic proteins such as VEGF. In the present study, we used methylotrophic yeast Pichia pastoris as the eukaryotic expression platform for the expression of chimeric HFBIIVEGF-A165. We investigated the effects of various chemical additives in the culture medium on yeast cell growth and chimeric HFBII-VEGFA165 production to achieve high concentrations of the recombinant target protein. 2. Materials and methods 2.1. Strains, media, and culture conditions Wild type of Pichia pastoris X-33 (Invitrogen) was used as the host cell to express recombinant HFBII-VEGF-A165 chimera. The plasmid pPICZA was used to construct the methanol inducible HFBII-VEGF-A165 chimera expression vector. Cloning steps were performed in E. coli DH5-α. The culture media contained YPD liquid or agar plate (yeast extract 10 g.L−1, peptone 20 g.L−1, dextrose 20 g.L−1, and agar 20 g.L−1), BMGY (yeast extract 10 g.L−1, peptone 20 g.L−1, potassium phosphate 100 mM and pH 6.0, YNB 13.4 g.L−1, biotin 0.4 mg.L−1, and 2% glycerol), and BMMY (same as BMGY with 0.5% methanol in place of glycerol), FM22 (KH2PO4 42.9 g.L−1, (NH4)2SO4 5 g.L−1, CaSO4·2H2O 1 g.L−1, K2SO4 14.3 g.L−1, MgSO4·7H2O 11.7 g.L−1, and with 2% glycerol and 0.5% methanol in growth and induction phases, respectively). Of Pichia trace minerals 4 (PTM4), 4.35mL was added per liter of the FM22 medium [30]. All experiments were done in 250 mL Erlenmeyer flasks with a working medium volume of 50 mL. Inoculation into all flasks was done such that the initial optical density at 600 nm was the same for all experiments. Yeast cells were cultivated at 200 rpm, and 30 °C for growth or 28 °C for recombinant protein expression. After 48 h of cultivation in the glycerol containing medium, the culture was centrifuged for 5 min at 1000g, and the medium was substituted with fresh inductive medium containing 0.5% pure methanol to initiate the recombinant protein production. The inductive condition was maintained for 96 h with the addition of 1% methanol every 24 h [31]. 2.2. Constructions of recombinant expression plasmid and cloning A synthetic gene encoding the sequences of HFBII from T. reesei (with its native signal sequence), which was fused upstream of the human VEGF-A165 (without signal sequence) via a (G3S)2 linker, was synthesized by Shanghai Generay Biotech Co. with optimal codon usage for Pichai pastoris. The HFBII-(G3S)2-VEGF-A165 construct was cloned using the EcoRI and KpnI restriction enzymes in the pPICZA plasmid under the control of AOX1 promoter and with HFBII signal sequence for protein secretion. The recombinant plasmid was transformed into competent E. coli DH5α using heat-shock method (90 s at 42 °C). Resistance to zeocin™ (25 μg.mL−1) was used to select
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for potential positive clones. The correct construction of recombinant plasmid was confirmed by PCR using HFBII and VEGF-A165 specific primers and sequencing. After propagation in the E. coli, the recombinant plasmid was linearized using SacI and electroporated to the fresh competent Pichia pastoris, as described previously [32]. Transformants were selected on YPD plates containing 100 μg.mL−1 zeocin™ and screened for the insert at AOX1 locus by PCR using AOX1 promoter and terminator primers and target gene-specific primers. Verified positive clones were used for subsequent analysis. 2.3. Separation of HFBII-VEGF-A165 chimera from the culture supernatant ATPS system was used to capture and concentrate the secretory HFBII fusion VEGF-A165 from the supernatant without the use of extra chromatographic column steps. Triton X-114 was used as the nonionic surfactant to perform phase separation at room temperature. The ATPS purification was done according to a previously described method with some modifications [33]. Briefly, the culture medium was centrifuged for 5 min at 1000g and in room temperature to precipitate the yeast cells and separate the supernatant containing the secreted recombinant VEGF-A165 fused to HFBII. Surfactant concentration has a major effect on protein purification quality and quantity in ATPS. Thus, different volumes of Triton X-114 were mixed with the supernatant to reach final concentrations of 1%, 2%, and 4% (w/v). The solutions were mixed completely using a vortex. The mixture was poured into a separation funnel and incubated for 15 min at 28 °C for phase separation. The hydrophobin fusion protein was concentrated within the micellar structure of surfactant and partitioned into the surfactant-rich lower phase, whereas other proteins primarily remained in the upper aqueous phase. The funnel tap was opened carefully and the lower phase was drained into a 50 mL conical tube. To extract the Triton X-114 from the solution containing the target protein, isobutyl alcohol was added to the collected phase in 10× volumes of initial surfactant and mixed by rolling the conical gently for b2 min. The phases were separated for 10 min and the bottom aqueous phase, containing the HFBII fusion target protein, was collected and centrifuged at 5000g for 5 min. The lower phase was separated from the upper phase and the precipitant. The remaining isobutyl alcohol was isolated from the target protein solution using centrifugal ultrafiltration (Corning® Spin-X UF6, 5000 MWCO). The resulting solution contained the HFBII fusion VEGF-A165. The final upper and lower phases of ATPS were analyzed with SDS-PAGE. 2.4. Binding activity of purified HFBII-VEGF-A165 to immobilized Ranibizumab Ranibizumab is a recombinant humanized IgG1 kappa monoclonal antibody fragment that binds to the receptor binding site of the active form of VEGF-A165 molecules and prevents their binding to VEGF receptors on endothelial cells [34]. An electrochemical design was used to assess the binding activity of VEGF portion of purified recombinant HFBIIVEGF-A165. Real-time binding study of purified recombinant HFBIIVEGF-A165 was monitored by an electrochemical biosensor composed of reduced graphene oxide (RGO) and gold nanoparticles (AuNPs), according to a previously described method [35]. Of the Ranibizumab solution, 20 ng/mL (Lucentis™, Genentech) was immobilized at the surface of electrode via covalent bonding. Electrochemical measurements were carried out using IviumStat XR (a potentiostat/galvanostat with frequency response analyzer) and cyclic voltammetry (CV) test was used to describe the performance of materials. 2.5. Effects of chemical additives on HFBII-VEGF-A165 production Effects of several additives in a chemically defined medium FM22 were evaluated on yeast cell growth and expression of recombinant HFBII-VEGF-A165 protein. The additives were PS, Triton X-100, cobalt, PBA, DMSO, and MgSO4. All the additives were used at the beginning
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Table 1 Concentration ranges for selected additives. #
Chemical additives
Concentration
References
1 2 3 4
PS Triton X-100 Cobalt 4-Phenylbutyric acid Dimethyl sulfoxide MgSO4
0.05, 0.1, 0.3, 0.6, 1 (% v/v) 0.5, 1, 1.5, 2 (% v/v) 10, 30, 60, 100 (μM) 30, 50, 100 (μM)
[47] [50] [51] [52,53]
0.5, 1, 1.5, 2 (% v/v) 4.5, 11.7, 17, 25, 32 (g.L−1)
[54] [51,54]
5 6
of the induction phase. Different concentrations of each additive were chosen based on the literature and our preliminary experiments to determine optimum concentration ranges (Table 1). Each experiment was performed in triplicate. The effect of each additive on the production of the target protein was quantified using ELISA. Densities of yeast cultures were calculated by measuring the OD at 600 nm. Interaction effects of chemical reagents with a positive response in the target protein expression were studied in the next step of media optimization using Response Surface Methodology (RSM). 2.6. Experimental design and data analysis The RSM based on Central Composite Design (CCD) was used to evaluate and optimize effects of additives concentrations, as independent variables, on our target protein expression as the main response function. The interaction effects and optimal parameters were obtained using Design-Expert software, version 10. Ranges and levels of independent variables are listed in Table S1 (Supplementary data). A total of 20 experiments with six central points were sufficient to calculate the coefficients of the second-order polynomial regression model for the three variables. The significance of the independent variables and their interactions were tested using analysis of variance (ANOVA) with a 92% confidence level.
All samples were diluted equally and assayed in triplicate. Standard VEGF supplied in the kit and the medium of non-recombinant Pichia pastoris were used as positive and negative controls, respectively. 3. Results 3.1. HFBII-VEGF-A165 expression and purification Fig. 1a shows a schematic representation of the genetic construct encoding for recombinant HFB-VEGF-A165. We used a positive recombinant yeast clone – verified by sequencing – to express the target protein, and the supernatant of the culture medium to evaluate the protein expression and purification. A specific ELISA assay confirmed the expression of recombinant VEGF-A165 in the culture medium and the SDSPAGE assay showed a band in ~20 kD for HFBII-VEGF-A165. The ATPS effectively separated the chimeric protein through its hydrophobic counterparts. Surfactant concentration had a significant effect on the separation. That is, lower concentrations of Triton X-114 led to more concentrated and purified HFBII-VEGF-A165 (Fig. 1b). 3.2. VEGF-A165 binding study We recorded voltammetric responses before and after addition of purified recombinant HFBII-VEGF-A165 to biosensor cell. As shown in Fig. 2, each CV curve has a redox peak that related to the electrochemical reaction of Fe+3/+2 ions. After immobilization of the antibody (Ranibizumab) on the surface of the electrode, the electrochemical active surface area decreased. Addition of HFBII-VEGF-A165 led to a larger decrease in surface area due to its interactions with the antibody and hindering the electron transfer rate of Fe+3/+2 ions and attenuating redox peaks. Furthermore, the fabricated biosensor revealed an excellent selectivity for the target protein compared with HFBII protein alone and bovine serum albumin (BSA) (data not shown). 3.3. Effects of additives on chimeric HFBII-VEGF-A165 expression and yeast cell growth
2.7. ELISA Culture supernatants from the last day of induction were subjected to ELISA to quantify the secreted recombinant HFBII-VEGF-A165 chimera. The experiments were done using VEGF Duo Set ELISA kit (DY293B, R&D systems), according to the manufacturer's instructions.
Supplementing the culture media with PS 20, PBA, and MgSO4 in selected concentration ranges had major effects on the secretory chimeric HFBII-VEGF-A165 production. Using the PS 20 additive in a 0.05–0.6% concentration range increased the production of chimeric HFBII-VEGFA165 significantly compared to the negative control condition (Fig. 3a).
Fig. 1. a) The map of recombinant plasmid pPICZA-HFBII-VEGF-A165. b) HFBII-VEGF-A165 purification with SDS-PAGE. Lane 1) protein marker, lane 2) the supernatant containing recombinant HFBII-VEGF-A165, lane 3) the lower phase of ATPS purification with 1% surfactant, lane 4) the upper phase of ATPS purification with 1% surfactant, lane 5) the lower phase of ATPS purification with 2% surfactant, lane 6) the upper phase of ATPS purification with 2% surfactant, lane 7) the lower phase of ATPS purification with 4% surfactant, lane 8) the upper phase of ATPS purification with 4% surfactant. According to the results, using 1% surfactant leads to higher concentrated and purified recombinant HFBII-VEGF-A165 in the lower phase.
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Fig. 2. Electrochemical CV curves of electrode modification procedure for detection of VEGF-A165, in the presence of 0.1 M hexacyanoferrate solution and at the scan rate of 50 mV/s. Step-down reduction in electrochemical active surface area was detected after modification of electrode with RGO/AuNPs, immobilization of Ranibizumab (Ab) and addition of HFBII-VEGF-A165.
We obtained the largest protein content using 0.05% PS 20 that gave 319% more protein than the negative control medium without PS 20. Increasing concentration of PS 20 reduced production of the protein linearly. Increasing PS 20 concentration reduced protein production but positively associated with the growth rate of P. pastoris (Fig. 3a). PBA in the concentration range of 30–100 μM significantly increased the chimeric HFBII-VEGF-A165 production (Fig. 3b). The largest increase was 307% with 30 μM of PBA. PBA addition did not affect cell growth. Increasing concentrations of MgSO4 from 11.7 to 32.0 g.L−1 consistently increased the production of target protein up to 250% of the negative control, but without any effect on the yeast cell growth (Fig. 3c). The other three additives, Triton X-100, DMSO, and cobalt, had inhibitory effects on the recombinant chimeric HFBII-VEGF-A165 production and cell growth (Supplementary Fig. 1) and were excluded from further studies.
3.4. RSM Using the results of the optimization experiments based on evaluating the effect of each parameter individually, we selected concentrations of PS 20, PBA, and MgSO4 that increased the chimeric HFBIIVEGF-A165 production for a CCD experiment. Table 2 shows the layout of the orthogonal arrays used, the corresponding responses, and chimeric HFBII-VEGF-A165 and biomass production. The optimal condition to achieve maximal extracellular production of chimeric HFBIIVEGF-A165 was supplementing the culture with 80 μM PBA, 0.8% PS 20, and 8.7 g.L−1 MgSO4. We further confirmed this optimum condition experimentally. Compared to the original FM22 medium, the new medium with the optimum levels of chemical additives led to a nearly 4.5 folds increase in chimeric HFBII-VEGF-A165 production, but no significant increase in the yeast biomass (Fig. 4). ANOVA results for the quadratic equation are shown in Table S2 (Supplementary data) that presents the equation and actual relationship between the response and significant variables. The model shows that PBA and MgSO4 (AC) had the strongest effect (p b 0.05) on HFBII-VEGF-A165 production. PS 20 (B) and the quadratic terms AB and A2 had a significant effect (p b 0.05) on the yield of the target protein production. However, PBA, MgSO4, and other quadratic terms (BC, B2, and C2) did not have a significant effect.
Fig. 3. Effects of culture media supplementation with PS 20 (a), PBA (b), and MgSO4 (c) on the production of chimeric HFBII-VEGF-A165 and P. pastoris cell growth. Each experiment was repeated in triplicate and error bars represent standard deviation from the mean. Statistical differences versus the respective controls are shown as ****p b 0.0001, ***p b 0.001, **p b 0.01, and *p b 0.05.
Fig. 5 shows the 3D plots of combined effects of PS 20, PBA, and MgSO4 concentrations on the chimeric HFBII-VEGF-A165 production. In each panel, one of the parameters is constant at its mean level, whereas the other two parameters vary within their experimental ranges. The result suggests that the largest PS 20 content (1%) and 50 μM PBA generated the greatest effect on the production of chimeric HFBII-VEGFA165 (Fig. 5a). The best interaction effect of MgSO4 and PBA for production of the target protein was in the presence of the highest concentration of PBA and the lowest concentration of MgSO4 (Fig. 5b). A simultaneous increase in concentrations of PS 20 and MgSO4 increased the production of chimeric HFBII-VEGF-A165 (Fig. 5c). 4. Discussion Purification is a major bottleneck and a costly process in commercial production of recombinant proteins. Different techniques of chromatography are generally utilized for selective and high-resolution purification of recombinant proteins. Traditional chromatographic methods
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Table 2 Experimental layout of CCD arrays according to RSM method. The maximum chimeric HFBII-VEGF-A165 production was obtained in trial 13. Trial
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Variables
Responses
PBA (μM)
PS (%)
MgSO4 (g.L−1)
VEGF (μg.mL−1)
Biomass (g.L−1)
20 50 80 20 50 50 80 20 50 80 50 20 80 50 50 100 50 50 0 50
0.8 0.5 0.8 0.2 0.0 0.5 0.2 0.8 0.5 0.2 0.5 0.2 0.8 0.5 0.5 0.5 0.5 1.0 0.5 0.5
20.8 14.7 20.8 20.8 14.7 14.7 8.7 8.7 14.7 20.8 0.0 8.7 8.7 14.7 14.7 14.7 25.0 14.7 14.7 14.7
178.2 162 130.5 162.9 108 171 156.6 108 160.2 67.5 153 135 211.5 165.6 162 99 167.4 207.9 135 188.1
14.95 14.5 14.35 14.65 14.9 14.7 14.65 14.5 14.7 14.62 14.65 14.5 14.75 14.25 14 14.55 14.55 14 14.75 14.25
are expensive and time-consuming, and are usually suited for analytical purposes or purification of high-value proteins. Moreover, the strategies can be hardly scaled up for industrial applications [36,37]. We described a fast, efficient, and economical method for selective protein purification in ATPS, based on the use of HFBII as the purification tag in a fusion protein. We designed a chimera of VEGF-A165 with HFBII, constructed it, and successfully expressed it in Pichia pastoris. We used the HFBII counterpart for low-cost and convenient purification of the target protein (VEGF-A165) from the supernatant of cell culture. HFBI was previously used for protein expression in plants and recovery of the fusion proteins from leaf extracts using ATPS [26,38–40]. However, we used HFBII as the fusion tag for protein purification in the Pichia pastoris expression system for the first time. We successfully isolated this water-soluble hydrophobin segment with its non-hydrophobic counterpart (VEGFA165) and concentrated it using an ATPS with a nonionic surfactant, Triton X-114. The low cloud point of Triton X-114 (22 °C) makes it a suitable detergent for the separation of proteins. At temperatures above its cloud point, Triton X-114 helps separate the detergent and aqueous phases. During separation, the hydrophobic solutes are sequestered in the detergent phase and separated from other non-hydrophobic compounds. The concentration of surfactant plays a major role in the quality and quantity of the recovered protein. Generally, increasing detergent concentration enhances protein recovery but reduces the recovery yield and conversely, decreasing detergent concentration leads to
Fig. 5. 3D response surface plots for HFBII-VEGF-A165 production showing the interactions effects of (a) PS 20 and PBA; (b) PBA and MgSO4; and (c) PS 20 and MgSO4.
Fig. 4. Production of chimeric HFBII-VEGF-A165 and P. pastoris cell growth in the optimal medium compared to the original FM22 medium.
poorer recovery but higher protein concentration [41]. We used three concentrations of Triton X-114 (1%, 2%, and 4% w/v) for hydrophobin protein recovery and found that 1% w/v of Triton X-114 led to more concentrated purified HFBII-VEGF-A165 protein. This result is consistent with a previous study by Reuter et al., which showed the capture of HFBI-GFP fusion protein from tobacco BY-2 cell suspension extract using ATPS method with Triton X-114 surfactant [33]. This inexpensive system resulted in a high recovery yield of ~95% (with 1% Triton X-114)
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target protein in 30–45 min and resolved the need for time-consuming and costly chromatographic methods. We chimerized HFBII to VEGF-A165 using a flexible linker (G3S)2 to avoid the structural/functional interference of the two components but use the advantages of HFBII for VEGF-A165 application (such as protective effects of HFBII and its antioxidant activity). We presented a newly designed biosensor based on the interaction between VEGFA165 and an anti-VEGF antibody, Ranibizumab. The antibody inactivates VEGF-A165 by binding to its receptor-binding region. The CV assay result indicated interactions between Ranibizumab and VEGF-A165, suggesting that the receptor-binding site of VEGF-A165 is not covered by HFBII binding site and is accessible for interaction with its receptors on endothelial cells. Tests for the functionality of VEGF-A165 in cell culture systems and in vivo will be conducted in future studies. In the event that HFBII has a negative impact on the VEGF-A165 activity, the HFBII may need to be cleaved off by incorporating a cleavage site (for chemical or enzymatic cleavage) in the linker. In this case, a second round of ATPS purification for segregation of the HFBII in the surfactant phase can lead to purification of the target protein in the aqueous phase in a soluble form. Although optimizing the purification process is necessary, it is not sufficient to improve the production of a recombinant protein. Various other parameters need to be studied and optimized in all steps of production. To achieve high production of recombinant HFBII-VEGF-A165 chimera from Pichia pastoris, we investigated effects of six different medium additives and their interactions. We selected the additives based on results of previous studies (Table 1). Among the selected chemicals, Mg2+, PS 20, and PBA enhanced the production of recombinant HFBIIVEGF-A165. Mg2+ is an inorganic compound and an essential mineral nutrient in biological systems. Mg2+ binds to ATP results in biologically active MgATP, which plays a key role in cell metabolism. Mg2+ is the cofactor of many enzymes including those involved in DNA or RNA synthesis or conversion of ADP to ATP. It impacts activities of different pathways including those that regulate supply of amino acids constituting the target protein [42]. Previous studies showed that specific concentrations of Mg2+ enhance the production of serine alkaline protease and IL-2 in E. coli [43,44]. Therefore, optimizing Mg2+ concentration is a useful strategy to enhance the expression level of recombinant proteins. In our study, increasing Mg2+ concentration in the form of MgSO4 increased the recombinant chimeric HFBII-VEGF-A165 expression without any effect on cell growth. PS 20 and 80, also known as Tween 20 and 80, respectively, are amphipathic, nonionic surfactants widely used as stabilizers in protein formulation to prevent surface adsorption and protein aggregation [45]. Non-ionic surfactants alter the membrane fluidity and permeability, led to further protein secretion [46]. It was shown that supplying 0.1% PS 20 to cell culture medium enhanced the expression levels of recombinant human growth hormone in shake flask and pilot-scale bioreactor [47]. Our results indicated that supplementing culture medium with b1% of PS 20 significantly increased cell growth and recombinant protein production. A 0.05% concentration of PS 20 generated the best effect on cell growth. PBA is a hydrophobic compound and a chemical chaperone that enhances protein folding and stability in recombinant protein production and reduces protein misfolding through a variety of mechanisms. A general mechanism of action of hydrophobic chemical chaperones is protection of protein from aggregation by interacting with exposed hydrophobic regions of the unfolded protein [48,49]. Our results confirmed the positive effects of PBA, in concentrations lower than 50 μM, on the recombinant protein expression but without affecting cell growth. Other tested additives, including cobalt, DMSO, and Triton X100 had a negative impact on the production of the target protein and were excluded from subsequent experiments. After identifying the effective additives and their effective ranges, we investigated the interaction effects of the additives for further improvements in the recombinant chimeric HFBII-VEGF-A165 expression. The
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production of the target protein increased by 450% using the CCD design. As shown in our results, the best medium formulation for chimeric HFBII-VEGF-A165 production occurred with the addition of 80 μM PBA, 0.8% PS 20, and 8.7 g.L−1 MgSO4. According to this model, PS 20 and quadratic terms of PBA and MgSO4 had the strongest effects on the chimeric HFBII-VEGF-A165 production in the recombinant P. pastoris. 5. Conclusions We investigated the production of recombinant chimeric HFBIIVEGF-A165 in P. pastoris and evaluated it using chemical additives including PS 20, Triton X-100, cobalt, PBA, DMSO, and MgSO4. We found that supplementing recombinant P. pastoris culture medium with PS 20, PBA, and MgSO4 significantly improved the production of the secretory HFBII-VEGF-A165 up to 4.5 folds. Orthogonal experiments showed that the optimal condition to achieve the largest amounts of HFBIIVEGF-A165 was with supplementing the medium with 80 μM PBA, 0.8% PS 20, and 8.7 g.L−1 MgSO4. We successfully used the HFBII counterpart for purification and concentration of the target chimeric protein using an ATPS with nonionic surfactant Triton X-114. Our study offers a novel approach to conveniently and inexpensively purify and recover recombinant proteins. Supplementary data to this article can be found online at https://doi. org/10.1016/j.ijbiomac.2019.08.080. Declaration of competing interest The authors declare that they have no competing interests. Acknowledgments This work was supported by Shahid Beheshti University, G.C., research grant. NO. 600/159. The authors appreciate the helpful comments of Dr. Javad Shabani Shayeh, Dr. Yahya Sefidbakht, and Mr. Mehrab Pourmadad. References [1] S.J. Harper, D.O. Bates, VEGF-A splicing: the key to anti-angiogenic therapeutics? Nat. Rev. Cancer 8 (11) (2008) 880–887. [2] D.I. Holmes, I. Zachary, The vascular endothelial growth factor (VEGF) family: angiogenic factors in health and disease, Genome Biol. 6 (2) (2005) 209. [3] W. Zhao, S.A. McCallum, Z. Xiao, F. Zhang, R.J. Linhardt, Binding affinities of vascular endothelial growth factor (VEGF) for heparin-derived oligosaccharides, Biosci. Rep. 32 (1) (2012) 71–81. [4] R.E. Gast, S. Konig, K. Rose, K.B. Ferenz, J. Krieglstein, Binding of ATP to vascular endothelial growth factor isoform VEGF-A165 is essential for inducing proliferation of human umbilical vein endothelial cells, BMC Biochem. 12 (2011) 28. [5] R. Roskoski Jr., Vascular endothelial growth factor (VEGF) and VEGF receptor inhibitors in the treatment of renal cell carcinomas, Pharmacol. Res. 120 (2017) 116–132. [6] K. Hu, B.R. Olsen, The roles of vascular endothelial growth factor in bone repair and regeneration, Bone 91 (2016) 30–38. [7] K.E. Johnson, T.A. Wilgus, Vascular endothelial growth factor and angiogenesis in the regulation of cutaneous wound repair, Advances in wound care 3 (10) (2014) 647–661. [8] Y.Y. Liao, Z.Y. Chen, Y.X. Wang, Y. Lin, F. Yang, Q.L. Zhou, New progress in angiogenesis therapy of cardiovascular disease by ultrasound targeted microbubble destruction, Biomed. Res. Int. 2014 (2014), 872984. [9] S.A. Pizarro, J. Gunson, M.J. Field, R. Dinges, S. Khoo, M. Dalal, M. Lee, K.A. Kaleas, K. Moiseff, S. Garnick, D.E. Reilly, M.W. Laird, C.H. Schmelzer, High-yield expression of human vascular endothelial growth factor VEGF(165) in Escherichia coli and purification for therapeutic applications, Protein Expr. Purif. 72 (2) (2010) 184–193. [10] A. Taktak-BenAmar, M. Morjen, H. Ben Mabrouk, R. Abdelmaksoud-Dammak, M. Guerfali, N. Fourati-Masmoudi, N. Marrakchi, A. Gargouri, Expression, purification and functionality of bioactive recombinant human vascular endothelial growth factor VEGF165 in E. coli, AMB Express 7 (1) (2017) 33. [11] X.L. Wei, L. Lin, Y. Hou, X. Fu, J.Y. Zhang, Z.B. Mao, C.L. Yu, Construction of recombinant adenovirus co-expression vector carrying the human transforming growth factor-beta1 and vascular endothelial growth factor genes and its effect on anterior cruciate ligament fibroblasts, Chin. Med. J. 121 (15) (2008) 1426–1432. [12] S.B. Lee, J.S. Park, S. Lee, J. Park, S. Yu, H. Kim, D. Kim, T.H. Byun, K. Baek, Y.J. Ahn, J. Yoon, Overproduction of recombinant human VEGF (vascular endothelial growth factor) in Chinese hamster ovary cells, J. Microbiol. Biotechnol. 18 (1) (2008) 183–187.
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Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac
Enhancing chimeric hydrophobin II-vascular endothelial growth factor A165 expression in Pichia pastoris and its efficient purification using hydrophobin counterpart Sareh Arjmand a,⁎, Zahra Tavasoli a, Seyed Omid Ranaei Siadat a,⁎, Behanm Saeidi a, Hossein Tavana b a b
Protein Research Center, Shahid Beheshti University, G. C., Tehran, Iran Department of Biomedical Engineering, The University of Akron, Akron, OH 44325, USA
a r t i c l e
i n f o
Article history: Received 14 May 2019 Received in revised form 3 August 2019 Accepted 8 August 2019 Available online 09 August 2019 Keywords: Vascular endothelial growth factor Hydrophobin Pichia pastoris Media optimization Protein purification
a b s t r a c t We report cloning and expressing of recombinant human VEGF-A165, fused at the N-terminal with Hydrophobin II (HFBII) from Trichoderma reseei, in yeast Pichia pastoris. We validated the construct using SDS-PAGE and ELISA against VEGF-A165 and efficiently performed protein purification and enrichment based on HFBII counterpart and using an aqueous two-phase system (ATPS) with nonionic surfactant X-114. We studied the effects of various culture medium additives and interaction effects of positive factors to increase the recombinant HFBII-VEGFA165 production. Supplementing the Pichia pastoris cell culture medium with Mg2+, Polysorbate 20 (PS 20), and 4-phenylbutyrate (PBA) improved the expression of the chimeric protein. Orthogonal experiments showed that the optimal condition to achieve maximal HFBII-VEGF-A165 production was with the addition of PBA, PS 20, and MgSO4. Under this condition, the production of the target protein was 4.5 times more than that in the medium without the additives. Overall, our approach to produce chimeric HFBII-VEGF-A165 and selectively capture it in ATPS is promising for large-scale protein production without laborious downstream processing. © 2019 Elsevier B.V. All rights reserved.
1. Introduction VEGF-A is a dimeric, disulfide-linked glycoprotein with key functions in both physiologic and pathologic neovascularization and angiogenesis. VEGF-A triggers multiple downstream signaling pathways by conjugating with the endothelial cell surface receptors including VEGF receptors 1 and 2 (VEGFR1 and VEGFR2) and the co-receptor neuropilin 1. VEGF-A has widely-expressed and tissue-specific isoforms, which are generated by alternative splicing and have different binding affinities to the VEGF receptors [1]. VEGF-A165 is the most abundant isoform that has binding affinity to all VEGF receptors. It plays a pivotal role in vascular development during embryonic development and wound healing [2]. This isoform also binds to heparin and heparin sulfate and regulates multiple aspects of angiogenesis and vasculogenesis [3]. VEGF-A165 is involved in cancer progression and several VEGF-A165 inhibitors have been approved for clinical use [4,5]. Additionally, this protein is useful for clinical applications such as wound healing, bone formation during bone repair, and prevention and treatment of ischemic heart disease [6–8]. Recombinant production and purification of VEGF-A165 and ⁎ Corresponding authors at: Protein Research Center, Shahid Beheshti University, G. C., PO Box 1983969411, Tehran, Iran. E-mail addresses: [email protected] (S. Arjmand), [email protected] (S.O. Ranaei Siadat).
https://doi.org/10.1016/j.ijbiomac.2019.08.080 0141-8130/© 2019 Elsevier B.V. All rights reserved.
related isoforms in different eukaryotic and prokaryotic expression systems with yields from micrograms to grams have been reported [9–12]. However, there is still a need for studies to optimize different steps of protein expression and purification to maximize the production efficiency and develop an economic and efficient industrial-scale production process. Optimization of media composition and/or addition of chemical supplements to growth media are among the traditional strategies to address this need. Numerous chemical additives have been shown to increase recombinant protein stability, solubility, folding, and expression. Among them, non-detergent surfactants, chemical chaperones, metal ions, and organic compounds have been widely studied. Positive effects of chemical additives are usually observed in certain ranges depending on the method of choice [13–16]. Optimization of the purification process is also used to increase the yield of recombinant protein production. Using purification tags as the fusion partner greatly simplifies the purification, although potential effects on the structure and biological activity of the target protein and stimulating immunogenic responses are the major drawbacks. For therapeutic proteins, it is usually necessary to remove the tag, but this results in extra steps and greater expense of production [17]. In this study, we considered the hydrophobin as a non-immunogenic, coexpressed fusion partner of VEGF-A165 for protein purification. Hydrophobins are a family of small, surface active, self-assembling
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proteins, which are produced by filamentous fungi and play a crucial role during the fungal life cycle. Hydrophobins share a unique structure consisting of four intramolecular disulfide bridges and exposed hydrophobic patches that are responsible for their amphipathic property [18–20]. These unique properties make hydrophobins desirable for many bio-industrial applications [21–24]. Hydrophobins are divided into two classes based on solubility and elasticity of their films: class I such as EAS from Neurospora crassa, and class II such as HFBI and HBFII from Trichoderma ressei [25]. This study focuses on HFBII due to its high aqueous solubility and film elasticity. Hydrophobins have been used as a fusing self-assembled tag in the expression and purification of several recombinant proteins, usually in plants, with considerable advantages such as high selectivity, high yield, and ease of use in the purification process [26–28]. Hydrophobin fusion technology has relied mainly on HFBI. It was recently shown that HFBII has properties such as anti-oxidant activity and Angiotensin Converting Enzyme (ACE) inhibitory effects [29], making it a suitable candidate as a fusion partner for therapeutic proteins such as VEGF. In the present study, we used methylotrophic yeast Pichia pastoris as the eukaryotic expression platform for the expression of chimeric HFBIIVEGF-A165. We investigated the effects of various chemical additives in the culture medium on yeast cell growth and chimeric HFBII-VEGFA165 production to achieve high concentrations of the recombinant target protein. 2. Materials and methods 2.1. Strains, media, and culture conditions Wild type of Pichia pastoris X-33 (Invitrogen) was used as the host cell to express recombinant HFBII-VEGF-A165 chimera. The plasmid pPICZA was used to construct the methanol inducible HFBII-VEGF-A165 chimera expression vector. Cloning steps were performed in E. coli DH5-α. The culture media contained YPD liquid or agar plate (yeast extract 10 g.L−1, peptone 20 g.L−1, dextrose 20 g.L−1, and agar 20 g.L−1), BMGY (yeast extract 10 g.L−1, peptone 20 g.L−1, potassium phosphate 100 mM and pH 6.0, YNB 13.4 g.L−1, biotin 0.4 mg.L−1, and 2% glycerol), and BMMY (same as BMGY with 0.5% methanol in place of glycerol), FM22 (KH2PO4 42.9 g.L−1, (NH4)2SO4 5 g.L−1, CaSO4·2H2O 1 g.L−1, K2SO4 14.3 g.L−1, MgSO4·7H2O 11.7 g.L−1, and with 2% glycerol and 0.5% methanol in growth and induction phases, respectively). Of Pichia trace minerals 4 (PTM4), 4.35mL was added per liter of the FM22 medium [30]. All experiments were done in 250 mL Erlenmeyer flasks with a working medium volume of 50 mL. Inoculation into all flasks was done such that the initial optical density at 600 nm was the same for all experiments. Yeast cells were cultivated at 200 rpm, and 30 °C for growth or 28 °C for recombinant protein expression. After 48 h of cultivation in the glycerol containing medium, the culture was centrifuged for 5 min at 1000g, and the medium was substituted with fresh inductive medium containing 0.5% pure methanol to initiate the recombinant protein production. The inductive condition was maintained for 96 h with the addition of 1% methanol every 24 h [31]. 2.2. Constructions of recombinant expression plasmid and cloning A synthetic gene encoding the sequences of HFBII from T. reesei (with its native signal sequence), which was fused upstream of the human VEGF-A165 (without signal sequence) via a (G3S)2 linker, was synthesized by Shanghai Generay Biotech Co. with optimal codon usage for Pichai pastoris. The HFBII-(G3S)2-VEGF-A165 construct was cloned using the EcoRI and KpnI restriction enzymes in the pPICZA plasmid under the control of AOX1 promoter and with HFBII signal sequence for protein secretion. The recombinant plasmid was transformed into competent E. coli DH5α using heat-shock method (90 s at 42 °C). Resistance to zeocin™ (25 μg.mL−1) was used to select
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for potential positive clones. The correct construction of recombinant plasmid was confirmed by PCR using HFBII and VEGF-A165 specific primers and sequencing. After propagation in the E. coli, the recombinant plasmid was linearized using SacI and electroporated to the fresh competent Pichia pastoris, as described previously [32]. Transformants were selected on YPD plates containing 100 μg.mL−1 zeocin™ and screened for the insert at AOX1 locus by PCR using AOX1 promoter and terminator primers and target gene-specific primers. Verified positive clones were used for subsequent analysis. 2.3. Separation of HFBII-VEGF-A165 chimera from the culture supernatant ATPS system was used to capture and concentrate the secretory HFBII fusion VEGF-A165 from the supernatant without the use of extra chromatographic column steps. Triton X-114 was used as the nonionic surfactant to perform phase separation at room temperature. The ATPS purification was done according to a previously described method with some modifications [33]. Briefly, the culture medium was centrifuged for 5 min at 1000g and in room temperature to precipitate the yeast cells and separate the supernatant containing the secreted recombinant VEGF-A165 fused to HFBII. Surfactant concentration has a major effect on protein purification quality and quantity in ATPS. Thus, different volumes of Triton X-114 were mixed with the supernatant to reach final concentrations of 1%, 2%, and 4% (w/v). The solutions were mixed completely using a vortex. The mixture was poured into a separation funnel and incubated for 15 min at 28 °C for phase separation. The hydrophobin fusion protein was concentrated within the micellar structure of surfactant and partitioned into the surfactant-rich lower phase, whereas other proteins primarily remained in the upper aqueous phase. The funnel tap was opened carefully and the lower phase was drained into a 50 mL conical tube. To extract the Triton X-114 from the solution containing the target protein, isobutyl alcohol was added to the collected phase in 10× volumes of initial surfactant and mixed by rolling the conical gently for b2 min. The phases were separated for 10 min and the bottom aqueous phase, containing the HFBII fusion target protein, was collected and centrifuged at 5000g for 5 min. The lower phase was separated from the upper phase and the precipitant. The remaining isobutyl alcohol was isolated from the target protein solution using centrifugal ultrafiltration (Corning® Spin-X UF6, 5000 MWCO). The resulting solution contained the HFBII fusion VEGF-A165. The final upper and lower phases of ATPS were analyzed with SDS-PAGE. 2.4. Binding activity of purified HFBII-VEGF-A165 to immobilized Ranibizumab Ranibizumab is a recombinant humanized IgG1 kappa monoclonal antibody fragment that binds to the receptor binding site of the active form of VEGF-A165 molecules and prevents their binding to VEGF receptors on endothelial cells [34]. An electrochemical design was used to assess the binding activity of VEGF portion of purified recombinant HFBIIVEGF-A165. Real-time binding study of purified recombinant HFBIIVEGF-A165 was monitored by an electrochemical biosensor composed of reduced graphene oxide (RGO) and gold nanoparticles (AuNPs), according to a previously described method [35]. Of the Ranibizumab solution, 20 ng/mL (Lucentis™, Genentech) was immobilized at the surface of electrode via covalent bonding. Electrochemical measurements were carried out using IviumStat XR (a potentiostat/galvanostat with frequency response analyzer) and cyclic voltammetry (CV) test was used to describe the performance of materials. 2.5. Effects of chemical additives on HFBII-VEGF-A165 production Effects of several additives in a chemically defined medium FM22 were evaluated on yeast cell growth and expression of recombinant HFBII-VEGF-A165 protein. The additives were PS, Triton X-100, cobalt, PBA, DMSO, and MgSO4. All the additives were used at the beginning
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Table 1 Concentration ranges for selected additives. #
Chemical additives
Concentration
References
1 2 3 4
PS Triton X-100 Cobalt 4-Phenylbutyric acid Dimethyl sulfoxide MgSO4
0.05, 0.1, 0.3, 0.6, 1 (% v/v) 0.5, 1, 1.5, 2 (% v/v) 10, 30, 60, 100 (μM) 30, 50, 100 (μM)
[47] [50] [51] [52,53]
0.5, 1, 1.5, 2 (% v/v) 4.5, 11.7, 17, 25, 32 (g.L−1)
[54] [51,54]
5 6
of the induction phase. Different concentrations of each additive were chosen based on the literature and our preliminary experiments to determine optimum concentration ranges (Table 1). Each experiment was performed in triplicate. The effect of each additive on the production of the target protein was quantified using ELISA. Densities of yeast cultures were calculated by measuring the OD at 600 nm. Interaction effects of chemical reagents with a positive response in the target protein expression were studied in the next step of media optimization using Response Surface Methodology (RSM). 2.6. Experimental design and data analysis The RSM based on Central Composite Design (CCD) was used to evaluate and optimize effects of additives concentrations, as independent variables, on our target protein expression as the main response function. The interaction effects and optimal parameters were obtained using Design-Expert software, version 10. Ranges and levels of independent variables are listed in Table S1 (Supplementary data). A total of 20 experiments with six central points were sufficient to calculate the coefficients of the second-order polynomial regression model for the three variables. The significance of the independent variables and their interactions were tested using analysis of variance (ANOVA) with a 92% confidence level.
All samples were diluted equally and assayed in triplicate. Standard VEGF supplied in the kit and the medium of non-recombinant Pichia pastoris were used as positive and negative controls, respectively. 3. Results 3.1. HFBII-VEGF-A165 expression and purification Fig. 1a shows a schematic representation of the genetic construct encoding for recombinant HFB-VEGF-A165. We used a positive recombinant yeast clone – verified by sequencing – to express the target protein, and the supernatant of the culture medium to evaluate the protein expression and purification. A specific ELISA assay confirmed the expression of recombinant VEGF-A165 in the culture medium and the SDSPAGE assay showed a band in ~20 kD for HFBII-VEGF-A165. The ATPS effectively separated the chimeric protein through its hydrophobic counterparts. Surfactant concentration had a significant effect on the separation. That is, lower concentrations of Triton X-114 led to more concentrated and purified HFBII-VEGF-A165 (Fig. 1b). 3.2. VEGF-A165 binding study We recorded voltammetric responses before and after addition of purified recombinant HFBII-VEGF-A165 to biosensor cell. As shown in Fig. 2, each CV curve has a redox peak that related to the electrochemical reaction of Fe+3/+2 ions. After immobilization of the antibody (Ranibizumab) on the surface of the electrode, the electrochemical active surface area decreased. Addition of HFBII-VEGF-A165 led to a larger decrease in surface area due to its interactions with the antibody and hindering the electron transfer rate of Fe+3/+2 ions and attenuating redox peaks. Furthermore, the fabricated biosensor revealed an excellent selectivity for the target protein compared with HFBII protein alone and bovine serum albumin (BSA) (data not shown). 3.3. Effects of additives on chimeric HFBII-VEGF-A165 expression and yeast cell growth
2.7. ELISA Culture supernatants from the last day of induction were subjected to ELISA to quantify the secreted recombinant HFBII-VEGF-A165 chimera. The experiments were done using VEGF Duo Set ELISA kit (DY293B, R&D systems), according to the manufacturer's instructions.
Supplementing the culture media with PS 20, PBA, and MgSO4 in selected concentration ranges had major effects on the secretory chimeric HFBII-VEGF-A165 production. Using the PS 20 additive in a 0.05–0.6% concentration range increased the production of chimeric HFBII-VEGFA165 significantly compared to the negative control condition (Fig. 3a).
Fig. 1. a) The map of recombinant plasmid pPICZA-HFBII-VEGF-A165. b) HFBII-VEGF-A165 purification with SDS-PAGE. Lane 1) protein marker, lane 2) the supernatant containing recombinant HFBII-VEGF-A165, lane 3) the lower phase of ATPS purification with 1% surfactant, lane 4) the upper phase of ATPS purification with 1% surfactant, lane 5) the lower phase of ATPS purification with 2% surfactant, lane 6) the upper phase of ATPS purification with 2% surfactant, lane 7) the lower phase of ATPS purification with 4% surfactant, lane 8) the upper phase of ATPS purification with 4% surfactant. According to the results, using 1% surfactant leads to higher concentrated and purified recombinant HFBII-VEGF-A165 in the lower phase.
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Fig. 2. Electrochemical CV curves of electrode modification procedure for detection of VEGF-A165, in the presence of 0.1 M hexacyanoferrate solution and at the scan rate of 50 mV/s. Step-down reduction in electrochemical active surface area was detected after modification of electrode with RGO/AuNPs, immobilization of Ranibizumab (Ab) and addition of HFBII-VEGF-A165.
We obtained the largest protein content using 0.05% PS 20 that gave 319% more protein than the negative control medium without PS 20. Increasing concentration of PS 20 reduced production of the protein linearly. Increasing PS 20 concentration reduced protein production but positively associated with the growth rate of P. pastoris (Fig. 3a). PBA in the concentration range of 30–100 μM significantly increased the chimeric HFBII-VEGF-A165 production (Fig. 3b). The largest increase was 307% with 30 μM of PBA. PBA addition did not affect cell growth. Increasing concentrations of MgSO4 from 11.7 to 32.0 g.L−1 consistently increased the production of target protein up to 250% of the negative control, but without any effect on the yeast cell growth (Fig. 3c). The other three additives, Triton X-100, DMSO, and cobalt, had inhibitory effects on the recombinant chimeric HFBII-VEGF-A165 production and cell growth (Supplementary Fig. 1) and were excluded from further studies.
3.4. RSM Using the results of the optimization experiments based on evaluating the effect of each parameter individually, we selected concentrations of PS 20, PBA, and MgSO4 that increased the chimeric HFBIIVEGF-A165 production for a CCD experiment. Table 2 shows the layout of the orthogonal arrays used, the corresponding responses, and chimeric HFBII-VEGF-A165 and biomass production. The optimal condition to achieve maximal extracellular production of chimeric HFBIIVEGF-A165 was supplementing the culture with 80 μM PBA, 0.8% PS 20, and 8.7 g.L−1 MgSO4. We further confirmed this optimum condition experimentally. Compared to the original FM22 medium, the new medium with the optimum levels of chemical additives led to a nearly 4.5 folds increase in chimeric HFBII-VEGF-A165 production, but no significant increase in the yeast biomass (Fig. 4). ANOVA results for the quadratic equation are shown in Table S2 (Supplementary data) that presents the equation and actual relationship between the response and significant variables. The model shows that PBA and MgSO4 (AC) had the strongest effect (p b 0.05) on HFBII-VEGF-A165 production. PS 20 (B) and the quadratic terms AB and A2 had a significant effect (p b 0.05) on the yield of the target protein production. However, PBA, MgSO4, and other quadratic terms (BC, B2, and C2) did not have a significant effect.
Fig. 3. Effects of culture media supplementation with PS 20 (a), PBA (b), and MgSO4 (c) on the production of chimeric HFBII-VEGF-A165 and P. pastoris cell growth. Each experiment was repeated in triplicate and error bars represent standard deviation from the mean. Statistical differences versus the respective controls are shown as ****p b 0.0001, ***p b 0.001, **p b 0.01, and *p b 0.05.
Fig. 5 shows the 3D plots of combined effects of PS 20, PBA, and MgSO4 concentrations on the chimeric HFBII-VEGF-A165 production. In each panel, one of the parameters is constant at its mean level, whereas the other two parameters vary within their experimental ranges. The result suggests that the largest PS 20 content (1%) and 50 μM PBA generated the greatest effect on the production of chimeric HFBII-VEGFA165 (Fig. 5a). The best interaction effect of MgSO4 and PBA for production of the target protein was in the presence of the highest concentration of PBA and the lowest concentration of MgSO4 (Fig. 5b). A simultaneous increase in concentrations of PS 20 and MgSO4 increased the production of chimeric HFBII-VEGF-A165 (Fig. 5c). 4. Discussion Purification is a major bottleneck and a costly process in commercial production of recombinant proteins. Different techniques of chromatography are generally utilized for selective and high-resolution purification of recombinant proteins. Traditional chromatographic methods
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Table 2 Experimental layout of CCD arrays according to RSM method. The maximum chimeric HFBII-VEGF-A165 production was obtained in trial 13. Trial
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Variables
Responses
PBA (μM)
PS (%)
MgSO4 (g.L−1)
VEGF (μg.mL−1)
Biomass (g.L−1)
20 50 80 20 50 50 80 20 50 80 50 20 80 50 50 100 50 50 0 50
0.8 0.5 0.8 0.2 0.0 0.5 0.2 0.8 0.5 0.2 0.5 0.2 0.8 0.5 0.5 0.5 0.5 1.0 0.5 0.5
20.8 14.7 20.8 20.8 14.7 14.7 8.7 8.7 14.7 20.8 0.0 8.7 8.7 14.7 14.7 14.7 25.0 14.7 14.7 14.7
178.2 162 130.5 162.9 108 171 156.6 108 160.2 67.5 153 135 211.5 165.6 162 99 167.4 207.9 135 188.1
14.95 14.5 14.35 14.65 14.9 14.7 14.65 14.5 14.7 14.62 14.65 14.5 14.75 14.25 14 14.55 14.55 14 14.75 14.25
are expensive and time-consuming, and are usually suited for analytical purposes or purification of high-value proteins. Moreover, the strategies can be hardly scaled up for industrial applications [36,37]. We described a fast, efficient, and economical method for selective protein purification in ATPS, based on the use of HFBII as the purification tag in a fusion protein. We designed a chimera of VEGF-A165 with HFBII, constructed it, and successfully expressed it in Pichia pastoris. We used the HFBII counterpart for low-cost and convenient purification of the target protein (VEGF-A165) from the supernatant of cell culture. HFBI was previously used for protein expression in plants and recovery of the fusion proteins from leaf extracts using ATPS [26,38–40]. However, we used HFBII as the fusion tag for protein purification in the Pichia pastoris expression system for the first time. We successfully isolated this water-soluble hydrophobin segment with its non-hydrophobic counterpart (VEGFA165) and concentrated it using an ATPS with a nonionic surfactant, Triton X-114. The low cloud point of Triton X-114 (22 °C) makes it a suitable detergent for the separation of proteins. At temperatures above its cloud point, Triton X-114 helps separate the detergent and aqueous phases. During separation, the hydrophobic solutes are sequestered in the detergent phase and separated from other non-hydrophobic compounds. The concentration of surfactant plays a major role in the quality and quantity of the recovered protein. Generally, increasing detergent concentration enhances protein recovery but reduces the recovery yield and conversely, decreasing detergent concentration leads to
Fig. 5. 3D response surface plots for HFBII-VEGF-A165 production showing the interactions effects of (a) PS 20 and PBA; (b) PBA and MgSO4; and (c) PS 20 and MgSO4.
Fig. 4. Production of chimeric HFBII-VEGF-A165 and P. pastoris cell growth in the optimal medium compared to the original FM22 medium.
poorer recovery but higher protein concentration [41]. We used three concentrations of Triton X-114 (1%, 2%, and 4% w/v) for hydrophobin protein recovery and found that 1% w/v of Triton X-114 led to more concentrated purified HFBII-VEGF-A165 protein. This result is consistent with a previous study by Reuter et al., which showed the capture of HFBI-GFP fusion protein from tobacco BY-2 cell suspension extract using ATPS method with Triton X-114 surfactant [33]. This inexpensive system resulted in a high recovery yield of ~95% (with 1% Triton X-114)
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target protein in 30–45 min and resolved the need for time-consuming and costly chromatographic methods. We chimerized HFBII to VEGF-A165 using a flexible linker (G3S)2 to avoid the structural/functional interference of the two components but use the advantages of HFBII for VEGF-A165 application (such as protective effects of HFBII and its antioxidant activity). We presented a newly designed biosensor based on the interaction between VEGFA165 and an anti-VEGF antibody, Ranibizumab. The antibody inactivates VEGF-A165 by binding to its receptor-binding region. The CV assay result indicated interactions between Ranibizumab and VEGF-A165, suggesting that the receptor-binding site of VEGF-A165 is not covered by HFBII binding site and is accessible for interaction with its receptors on endothelial cells. Tests for the functionality of VEGF-A165 in cell culture systems and in vivo will be conducted in future studies. In the event that HFBII has a negative impact on the VEGF-A165 activity, the HFBII may need to be cleaved off by incorporating a cleavage site (for chemical or enzymatic cleavage) in the linker. In this case, a second round of ATPS purification for segregation of the HFBII in the surfactant phase can lead to purification of the target protein in the aqueous phase in a soluble form. Although optimizing the purification process is necessary, it is not sufficient to improve the production of a recombinant protein. Various other parameters need to be studied and optimized in all steps of production. To achieve high production of recombinant HFBII-VEGF-A165 chimera from Pichia pastoris, we investigated effects of six different medium additives and their interactions. We selected the additives based on results of previous studies (Table 1). Among the selected chemicals, Mg2+, PS 20, and PBA enhanced the production of recombinant HFBIIVEGF-A165. Mg2+ is an inorganic compound and an essential mineral nutrient in biological systems. Mg2+ binds to ATP results in biologically active MgATP, which plays a key role in cell metabolism. Mg2+ is the cofactor of many enzymes including those involved in DNA or RNA synthesis or conversion of ADP to ATP. It impacts activities of different pathways including those that regulate supply of amino acids constituting the target protein [42]. Previous studies showed that specific concentrations of Mg2+ enhance the production of serine alkaline protease and IL-2 in E. coli [43,44]. Therefore, optimizing Mg2+ concentration is a useful strategy to enhance the expression level of recombinant proteins. In our study, increasing Mg2+ concentration in the form of MgSO4 increased the recombinant chimeric HFBII-VEGF-A165 expression without any effect on cell growth. PS 20 and 80, also known as Tween 20 and 80, respectively, are amphipathic, nonionic surfactants widely used as stabilizers in protein formulation to prevent surface adsorption and protein aggregation [45]. Non-ionic surfactants alter the membrane fluidity and permeability, led to further protein secretion [46]. It was shown that supplying 0.1% PS 20 to cell culture medium enhanced the expression levels of recombinant human growth hormone in shake flask and pilot-scale bioreactor [47]. Our results indicated that supplementing culture medium with b1% of PS 20 significantly increased cell growth and recombinant protein production. A 0.05% concentration of PS 20 generated the best effect on cell growth. PBA is a hydrophobic compound and a chemical chaperone that enhances protein folding and stability in recombinant protein production and reduces protein misfolding through a variety of mechanisms. A general mechanism of action of hydrophobic chemical chaperones is protection of protein from aggregation by interacting with exposed hydrophobic regions of the unfolded protein [48,49]. Our results confirmed the positive effects of PBA, in concentrations lower than 50 μM, on the recombinant protein expression but without affecting cell growth. Other tested additives, including cobalt, DMSO, and Triton X100 had a negative impact on the production of the target protein and were excluded from subsequent experiments. After identifying the effective additives and their effective ranges, we investigated the interaction effects of the additives for further improvements in the recombinant chimeric HFBII-VEGF-A165 expression. The
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production of the target protein increased by 450% using the CCD design. As shown in our results, the best medium formulation for chimeric HFBII-VEGF-A165 production occurred with the addition of 80 μM PBA, 0.8% PS 20, and 8.7 g.L−1 MgSO4. According to this model, PS 20 and quadratic terms of PBA and MgSO4 had the strongest effects on the chimeric HFBII-VEGF-A165 production in the recombinant P. pastoris. 5. Conclusions We investigated the production of recombinant chimeric HFBIIVEGF-A165 in P. pastoris and evaluated it using chemical additives including PS 20, Triton X-100, cobalt, PBA, DMSO, and MgSO4. We found that supplementing recombinant P. pastoris culture medium with PS 20, PBA, and MgSO4 significantly improved the production of the secretory HFBII-VEGF-A165 up to 4.5 folds. Orthogonal experiments showed that the optimal condition to achieve the largest amounts of HFBIIVEGF-A165 was with supplementing the medium with 80 μM PBA, 0.8% PS 20, and 8.7 g.L−1 MgSO4. We successfully used the HFBII counterpart for purification and concentration of the target chimeric protein using an ATPS with nonionic surfactant Triton X-114. Our study offers a novel approach to conveniently and inexpensively purify and recover recombinant proteins. Supplementary data to this article can be found online at https://doi. org/10.1016/j.ijbiomac.2019.08.080. Declaration of competing interest The authors declare that they have no competing interests. Acknowledgments This work was supported by Shahid Beheshti University, G.C., research grant. NO. 600/159. The authors appreciate the helpful comments of Dr. Javad Shabani Shayeh, Dr. Yahya Sefidbakht, and Mr. Mehrab Pourmadad. References [1] S.J. Harper, D.O. Bates, VEGF-A splicing: the key to anti-angiogenic therapeutics? Nat. Rev. Cancer 8 (11) (2008) 880–887. [2] D.I. Holmes, I. Zachary, The vascular endothelial growth factor (VEGF) family: angiogenic factors in health and disease, Genome Biol. 6 (2) (2005) 209. [3] W. Zhao, S.A. McCallum, Z. Xiao, F. Zhang, R.J. Linhardt, Binding affinities of vascular endothelial growth factor (VEGF) for heparin-derived oligosaccharides, Biosci. Rep. 32 (1) (2012) 71–81. [4] R.E. Gast, S. Konig, K. Rose, K.B. Ferenz, J. Krieglstein, Binding of ATP to vascular endothelial growth factor isoform VEGF-A165 is essential for inducing proliferation of human umbilical vein endothelial cells, BMC Biochem. 12 (2011) 28. [5] R. Roskoski Jr., Vascular endothelial growth factor (VEGF) and VEGF receptor inhibitors in the treatment of renal cell carcinomas, Pharmacol. Res. 120 (2017) 116–132. [6] K. Hu, B.R. Olsen, The roles of vascular endothelial growth factor in bone repair and regeneration, Bone 91 (2016) 30–38. [7] K.E. Johnson, T.A. Wilgus, Vascular endothelial growth factor and angiogenesis in the regulation of cutaneous wound repair, Advances in wound care 3 (10) (2014) 647–661. [8] Y.Y. Liao, Z.Y. Chen, Y.X. Wang, Y. Lin, F. Yang, Q.L. Zhou, New progress in angiogenesis therapy of cardiovascular disease by ultrasound targeted microbubble destruction, Biomed. Res. Int. 2014 (2014), 872984. [9] S.A. Pizarro, J. Gunson, M.J. Field, R. Dinges, S. Khoo, M. Dalal, M. Lee, K.A. Kaleas, K. Moiseff, S. Garnick, D.E. Reilly, M.W. Laird, C.H. Schmelzer, High-yield expression of human vascular endothelial growth factor VEGF(165) in Escherichia coli and purification for therapeutic applications, Protein Expr. Purif. 72 (2) (2010) 184–193. [10] A. Taktak-BenAmar, M. Morjen, H. Ben Mabrouk, R. Abdelmaksoud-Dammak, M. Guerfali, N. Fourati-Masmoudi, N. Marrakchi, A. Gargouri, Expression, purification and functionality of bioactive recombinant human vascular endothelial growth factor VEGF165 in E. coli, AMB Express 7 (1) (2017) 33. [11] X.L. Wei, L. Lin, Y. Hou, X. Fu, J.Y. Zhang, Z.B. Mao, C.L. Yu, Construction of recombinant adenovirus co-expression vector carrying the human transforming growth factor-beta1 and vascular endothelial growth factor genes and its effect on anterior cruciate ligament fibroblasts, Chin. Med. J. 121 (15) (2008) 1426–1432. [12] S.B. Lee, J.S. Park, S. Lee, J. Park, S. Yu, H. Kim, D. Kim, T.H. Byun, K. Baek, Y.J. Ahn, J. Yoon, Overproduction of recombinant human VEGF (vascular endothelial growth factor) in Chinese hamster ovary cells, J. Microbiol. Biotechnol. 18 (1) (2008) 183–187.
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