The purification of recombinant proteins from plant systems is usually challenged by plant proteins. This work provides a method to effectively extract and purify a secreted recombinant protein from the apoplast of Nicotiana benthamiana.
Plants are a newly developing eukaryotic expression system being explored to produce therapeutic proteins. Purification of recombinant proteins from plants is one of the most critical steps in the production process. Typically, proteins were purified from total soluble proteins (TSP), and the presence of miscellaneous intracellular proteins and cytochromes poses challenges for subsequent protein purification steps. Moreover, most therapeutic proteins like antigens and antibodies are secreted to obtain proper glycosylation, and the presence of incompletely modified proteins leads to inconsistent antigen or antibody structures. This work introduces a more effective method to obtain highly purified recombinant proteins from the plant apoplastic space. The recombinant Green fluorescent protein (GFP) is engineered to be secreted into the apoplast of Nicotiana benthamiana and is then extracted using an infiltration-centrifugation method. The GFP-His from the extracted apoplast is then purified by nickel affinity chromatography. In contrast to the traditional methods from TSP, purification from the apoplast produces highly purified recombinant proteins. This represents an important technological improvement for plant production systems.
Nowadays, various plant-produced recombinant therapeutic proteins are under study, including antibodies, vaccines, bioactive proteins, enzymes, and small polypeptides1,2,3,4,5,6. Plants are becoming an increasingly utilized platform for producing therapeutic proteins due to their safety, low cost, and ability to rapidly scale production7,8. Protein expression and modification in plant systems, along with protein purification, are critical factors determining the productivity of plant bioreactors9,10. Enhancing plant productivity and obtaining high-purity target proteins are core challenges facing plant-based production systems11,12.
The subcellular localization and modification of recombinant proteins depends on the specific signal peptide encoded at the N-terminus, which targets the protein to its final organelle destination during gene expression. Recombinant proteins are designed to localize to the apoplast, endoplasmic reticulum (ER), chloroplast, vacuole, or cytoplasm, based on their unique properties13. For antigens and antibodies, secreted proteins are preferred. Before secretion, proteins progress through ER and Golgi for correct folding and post-translational modifications14,15,16.
After production in plants, recombinant proteins must be purified from plant extracts. Typically, proteins are purified from total soluble plant extracts, which contain abundant intracellular proteins, misfolded and unmodified products, and cytochromes17. To remove impurities, plant extracts undergo extensive fractionation, including ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO)-specific affinity chromatography, phytate precipitation, polyethylene glycol precipitation, and adjustment of temperature and pH18,19. Such impurity-removal steps are laborious and can compromise protein quality.
The plant cell compartment beyond the plasma membrane is defined as the apoplast, encompassing the cell wall, intercellular spaces, and xylem20. The aqueous phase is commonly called apoplast washing fluid (AWF). AWF contains molecules secreted by cells to regulate numerous biological progresses like transport, cell wall metabolism, and stress responses21,22. In contrast to TSP, the molecular constituents of AWF are less complex. Extracting recombinant proteins from AWF may avoid contamination by intracellular proteins, misfolded products, and cytoplasmic remnants. Briefly, extraction lipid enters leaves through the stomata under negative pressure, and the AWF is collected by gentle centrifugation23. While isolation of AWF is widely employed to study the biological progress inside apoplast24,25,26,27, it has not been exploited in plant-based production systems.
Improving plant productivity and obtaining high purity target proteins are central challenges for plant production systems28. Typically, proteins are purified from total soluble plant extracts containing large amounts of intracellular proteins, misfolded and unmodified products, and cytochromes29, which require great costs for subsequent purification30. The use of AWF protein extraction methods for the extraction of exogenous recombinant proteins secreted into apoplast can effectively improve the homogeneity of recombinant proteins and reduce the cost of protein purification. Here, we use a signal peptide PR1a to enable exogenous protein secretion into the apoplast space and introduce a detailed method for recombinant protein purification from the AWF of N. benthamiana.
1. Preparation of N. benthamiana plants
2. Construction of the recombinant GFP-His
3. Production of GFP-His in N. benthamiana
4. Observing subcellular localization of GFP
5. Extracting GFP from plant
6. Purification of GFP-His with nickel (Ni) affinity chromatography
NOTE: Perform all steps at 4 °C.
7. Coomassie blue staining of proteins
8. Western blotting of proteins
9. Protein concentration assay
10. Protein quantification 31
GFP-His is targeted for secretion into the apoplastic space of N. benthamiana
GFP was cloned into the pCAMBIA1300 plasmid with an N-terminal PR1a signal peptide for secretion and a C-terminal His-tag for purification, generating p35s-PR1a-GFP-His (Figure 1A). The recombinant protein was transiently expressed in N. benthamiana and a 30 kDa band was detected by western blot using anti-His antibody at 3 days post agroinfiltration (Figure 1B). Plasmolysis is the treatment of leaves using a hypertonic solution to separate the cell wall from the cell membrane32. To determine whether GFP is located in the apoplast, we used 30% sucrose to make the leaves plasmolysis. Fluorescence microscopy illustrated the presence of GFP-His within the apoplast (Figure 1C). AWF proteins extracted by vacuum infiltration-centrifugation also contained GFP-His (Figure 1D). These results demonstrate the secretion of GFP-His into the AWF of N. benthamiana leaves, from which it can be extracted for downstream purification.
Protein purification from AWF
The efficacy of AWF extraction strongly influences the final GFP yield. Building on published AWF extraction protocols24,25,26,27, we tested various solutions to optimize the recovery of secreted GFP-His. Here, eight extraction solutions were examined: 25 mM Tris-HCl (pH 7.4), 100 mM NaH2PO4 (pH 4.5), 50 mM NaCl (pH 7.1), 40 mM KCl (pH 7.0), 20 mM CaCl2 (pH 5.7), 100 mM PB (pH 7.0), 10 mM MES (pH 5.6) and ddH2O (pH 7.0). PB extracted the greatest amount of AWF and GFP-His, exceeding other solutions by over 4x (Figure 2A,B). Comparing PB at varying pH revealed improved AWF and GFP-His extraction at neutral to mildly alkaline conditions (pH 6.0, 6.5, 7.0, 7.5, and 8.0; Figure 2C,D). Additionally, PB with concentrations of 50 mM, 100 mM, and 150 mM were compared for the extraction efficacy. Results indicated that 100 mM PB showed maximal efficacy for recovering both AWF and GFP-His (Figure 2E,F).
Based on these optimization experiments, 100 mM PB, pH 7.0, was selected for large-scale AWF extraction from N. benthamiana. Using this buffer, 495 µg of total AWF proteins per gram of fresh leaf material were recovered from the apoplast (Figure 2G). The extracted AWF contained 10 µg of GFP-His (Figure 2H), which corresponded to approximately 18% of the total GFP-His in the TSP (Figure 2I). Following Ni-affinity chromatography, GFP-His purity reached 84.3% from AWF extraction, substantially higher than the 44.9% purity achieved through total soluble protein extraction (Figure 2J).
Figure 1: GFP-His is targeted for secretion into the apoplast of N. benthamiana. (A) Schematic of PR1a-GFP-His construction in the pCAMBIA1300 plant expression vector. (B) Western blot detecting GFP-His production in N. benthamiana. Here,10 µL of protein samples were loaded and detected. The N. benthamiana leaves simultaneously injected with MMA were used as a negative control. The same volume of samples was examined and stained using plant large subunits as loading control (C) Confocal microscopy showing the subcellular localization of GFP-His (green). N. benthamiana leaves were plasmolyzed with 30% sucrose. White dashed lines indicate the plasma membrane. Red arrows denote GFP-His accumulation in the apoplast. (D) Western blot demonstrating the presence of GFP-His in the extracted apoplastic fluid. Here,10 µL of protein samples were loaded and detected. The N. benthamiana leaves simultaneously injected with MMA were used as a negative control. Abbreviations: 35s = 2x Cauliflower mosaic virus 35s promoter; PR1a = tobacco pathogenesis-related protein 1a signal peptide; His = 6x His tag; Nos = nopaline synthase terminator. BS = before plasmolysis; PS = post-plasmolysis. Please click here to view a larger version of this figure.
Figure 2: Optimization of the protein extraction method from AWF. (A-B) Extraction of proteins from AWF using different buffers. Eight solutions were tested for protein extraction from AWF: 25 mM Tris-HCl (pH 7.4), 100 mM NaH2PO4 (pH 4.5), 50 mM NaCl (pH 7.0), 40 mM KCl (pH 7.0), 20 mM CaCl2 (pH 5.7), 100 mM PB (pH 7.0), 10 mM MES (pH 5.6), and ddH2O (pH 7.0). The extraction efficiency was analyzed by SDS-PAGE (A), and GFP-His was detected by western blotting using anti-His antibody (B). (C-D) Extraction using PB at different pH values. PB with pH 6.0, 6.5, 7.0, 7.5, and 8.0 was used for extraction from AWF. The extraction efficiency was detected by SDS-PAGE (C), and GFP-His was detected by western blotting with anti-His antibody (D). (E-F) Extraction using PB at different concentrations. PB at 50 mM, 100 mM, and 150 mM were used for extraction from AWF. The extraction efficiency was analyzed by SDS-PAGE (E), and GFP-His was detected by western blotting with anti-His antibody (F). (G-H) Quantitative analysis of extracted proteins. Proteins were extracted using 100 mM PB (pH 7.0). The total soluble protein (G) and GFP-His (H) were quantified for statistical analysis. (I) Statistical analysis of the proportion of AWF GFP to total GFP. (J) Purification of GFP-His from total soluble proteins of N. benthamiana or AWF. Proteins were extracted using 100 mM PB (pH 7.0), and GFP-His was purified by Ni affinity chromatography. Purification was analyzed by SDS-PAGE. The rightmost lane is the 1 µg BSA standard. Please click here to view a larger version of this figure.
Using plants to produce therapeutic proteins has expanded quickly in recent years1,2,3,4,5,6. Protein-encoding genes are cloned into expression vectors and delivered into plant tissues via agroinfiltration. After production by plant cells, proteins are purified for downstream applications. Typically, recombinant proteins are purified from TSP extracts. However, abundant plant proteins and cytochromes released from plant cells during extraction can hinder purification33. On the one hand, plant cytochromes can clog separation columns, slowing processing17; on the other hand, highly abundant proteins like RuBisCO are difficult to remove, reducing purity18,19. These issues become more problematic when the target proteins are expressed at low levels. Purifying from AWF bypasses these issues, enabling higher purity (Figure 2J).
While AWF extraction has been widely used in plant biology24,25,26,27, it is rarely exploited in molecular farming pipelines. Here, we use a plant-made GFP model to demonstrate AWF-based protein purification. Extraction of GFP from the AWF was performed by adding a signal peptide to enable GFP secretion into the apoplast space. This method extracts proteins with higher homogeneity and reduces downstream protein processing costs. Several considerations merit future investigation for implementing AWF purification in plant bioreactors. First, efficient secretion requires the fusion of specific N-terminal signal peptides. Second, to preserve AWF quality, intact leaves must be processed rapidly after harvest without freezing, which releases intracellular contents. Third, PB effectively extracts AWF proteins34 but buffer pH and salt concentration should be tailored for individual recombinant proteins (Figure 2C-D). Fourth, we achieved purified protein from 200 g of N. benthamiana many times in laboratory conditions with stable purity and yield. To be scaled up for use in the factory, larger vessels and vacuum pumps and a more effective purification method are asked, and conditions such as vacuum duration, number of recoveries, etc., may need to be optimized.
This method is limited by the fact that exogenous proteins are secreted into the AWF, and proteins inside the plasma membrane are deserted (Figure 2H-I). It brings the benefit of getting higher purified proteins with correct modification but at a higher cost of yield. The major technical bottleneck for AWF-based purification is the improvement of the target protein level inside AWF. Coupling with strong promoters and optimized secretion systems will improve plant expression platforms for both high purity and greater yields. To overcome this bottleneck, a higher production system and a more effective secretion system are asked for in future work.
The authors have nothing to disclose.
This work is supported by the Youth Innovation Promotion Association CAS (2021084) and the Key Deployment Project Support Fund of the Three-Year Action Plan of the Institute of Microbiology, Chinese Academy of Sciences.
100 mesh nylon fine mesh yarn | Guangzhou Huayu Trading limited company | hy-230724-2 | For AWF protein extraction |
50 ml centrifuge tubes | Vazyme | TCF00150 | For centrifuge |
ClonExpress II one step cloning kit | Vazyme | C112-02 | For clone |
FastDigest Sac1 | NEB | R3156S | For clone |
FastDigest XbaI | NEB | FD0685 | For clone |
ImageJ 1.5.0 | / | / | For greyscale analysis |
Large filter | Thermo Fisher | NEST | For filtering |
Laser scanning confocal microscopy | Leica SP8 | For subcellular localization | |
Ni sepharose excel | Cytiva | 10339806 | For protein purification |
Precast protein plus gel | YEASEN | 36246ES10 | For SDS-PAGE |
Small filter | Nalgene | 1660045 | For filtering |
SuperSignal West Femto Substrate Trial Kit | Thermo Fisher | 34076 | For western blotting |
Triton X-100 | SCR | 30188928 | To configure the extraction buffer |
Type rotaryvang vacuum pump | Zhejiang taizhouqiujing vacuum pump limited company | 2XZ-2 | For vacuum infiltration |
Vertical mixer | Haimen Qilinbel Instrument Manufacturing limited company | BE-1200 | For mixing |
β-mercaptoethanol | SIGMA | BCBK8223V | To configure the extraction buffer |
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