Apoplast-Extraction Based Method to Improve the Purity of Plant Produced Recombinant Protein

Abstract

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.

Introduction

Nowadays, various plant-produced recombinant therapeutic proteins are under study, including antibodies, vaccines, bioactive proteins, enzymes, and small polypeptides^1,2,3,4,5,6. Plants are becoming an increasingly utilized platform for producing therapeutic proteins due to their safety, low cost, and ability to rapidly scale production^7,8. Protein expression and modification in plant systems, along with protein purification, are critical factors determining the productivity of plant bioreactors^9,10. Enhancing plant productivity and obtaining high-purity target proteins are core challenges facing plant-based production systems^11,12.

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 modifications^14,15,16.

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 pH^18,19. 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 responses^21,22. 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 apoplast^24,25,26,27, 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.

Protocol

1. Preparation of N. benthamiana plants

1. Place seedling blocks in a tray, add 1 L of water and soak for approximately 2 h until fully saturated.
2. Evenly sprinkle wild-type N. benthamiana seeds on the seedling blocks, cover with a lid, and germinate for 1 week. Grow N. benthamiana in a greenhouse with 18 h photoperiod, 24 °C, 45% relative humidity, and fertilize every 2 weeks.
3. Once the seeds have sprouted, use tweezers to gently remove a seedling, taking care not to cause damage to its roots. Dig a small pit in the center of a new tray, place the seedling in it, and cover the roots lightly with soil. Cover with a lid and continue to grow.
4. Grow till the seedlings have three leaves, with the lid on. Remove the lid when the 4^th true leaf emerges.
5. Allow plants to continue growing for two more weeks before agroinfiltration.

2. Construction of the recombinant GFP-His

1. Perform N. benthamiana codon optimization and synthesis of GFP and PR1a (MGFVLFSQLPSFLLVSTLLLFLVISHSCRAG) based on the gene sequences.
2. Carry out double digestion of pCAMBIA1300 vector using Xba I and Sac I enzymes (see Table of Materials).
3. Perform amplification of PR1a using upstream primers (TTGGAGAGAACACGGGGGACTCTAGAATGGGATT
   TGTTCTCTTTTC) and downstream primers (TCCTCG
   CCCTTGCTCACCATGTCGACCCCGGCACGGCAAGA
   GTGGG), amplification of GFP using upstream primers (CCCACTCTTGCCGTGCCGGGGTCGACATGGTGAG
   CAAGGGCGAGGA) and downstream primers (GGGGAAATTCGAGCTCTCAGTGGTGATGGTGGTG
   GTGAGATCTTCCGGACTTGT).
4. Using homologous recombination, clone PR1a, and GFP into the pCAMBIA1300 plant expression vector. After transferring into E. coli, select single clones and send them to the company for sequencing. Comparison of sequencing results and the right plasmid is the recombinant plasmid pCAMBIA1300-PR1a-GFP-His, referred to as p35s-PR1a-GFP-His.

3. Production of GFP-His in N. benthamiana

1. Add 1 µL of GFP plasmid into 50 µL of Agrobacterium receptor cells GV3101, mix gently and add to the electrode cup, remove after electrocution on the electroshocker.
2. Spread transformed Agrobacterium tumefaciens on an LB solid medium plate containing 50 µg/mL kanamycin (Kan) and 50 µg/mL rifampicin (Rif). Incubate inverted at 28 °C for 48 h. The pCAMBIA1300 vector is kan-resistant, and Agrobacterium is rif-resistant; only the correct Agrobacterium can grow on plates with kan and rif. The monoclonal clones were the positive transformants, named A. tumefaciens GV3101-p35s-PR1a-GFP-His (AtGPG).
3. Use a sterilized toothpick to pick up an appropriate amount of AtGPG and inoculate it into 4 mL of liquid LB with Kan and Rif. Culture at 28 °C, 220-300 rpm for 24 h.
4. Subculture bacteria 1:50 into fresh LB (50 µg/mL Kan, 10 mM 2-(N-Morpholino) ethanesulfonic acid (MES), 20 µM acetosyringone (AS)) and incubate at 28 °C, 220-300 rpm for 10-12 h.
5. Harvest bacteria by centrifugation at 1500 x g for 10 min and resuspend the pellet with 1 mL of MMA (10 mM MgCl₂, 10 mM MES pH 5.6, 100 µM AS).
6. Measure and adjust the optical density (OD) of AtGPG to OD₆₀₀=0.8 using MMA. Incubate the bacteria for another 2-4 h at room temperature. Draw up the bacterial solution with a needleless syringe, press it against the back of the leaves lightly, and give pressure to penetrate the solution into the leaves. Inject only the middle 3-4 leaves per plant.
7. Continue to grow N. benthamiana in the greenhouse for 3-4 days before harvesting leaves for protein extraction.

4. Observing subcellular localization of GFP

1. After 48 h of agroinfiltration, select a flat leaf and use a 6 mm hole punch to clamp the blade for punching.
2. Drip 30% sucrose solution onto a glass slide, place the leaf sample with the back facing upwards and cover it with a cover glass.
3. Set the excitation wavelength to 488 nm and detect the green fluorescence at 490-550 nm after magnification of 400x. Take the first photo once the slide is prepared and another one 10 min later.

5. Extracting GFP from plant

1. AWF extraction
   1. Collect the intact fresh N. benthamiana leaves expressing recombinant GFP at 3 days post agroinfiltration. Treat the leaf gently and maintain leaf integrity to avoid intracellular protein efflux. Use collected leaves for the next step immediately to avoid protein degradation.
   2. Rinse leaf blade with pre-cooled sterilized ddH₂O to remove debris.
   3. Weigh fresh leaves and submerge 20 g leaves (abaxial side down) in 100 mL of pre-cooled 100 mM phosphate buffer (PB 100 mM Na₂HPO₄ and 100 mM NaH₂PO₄ were separately configured and mixed at a ratio of 2.2:1 pH 7.0) in a 200 mL wide-mouth beaker. Cover leaves with 100 mesh nylon fine mesh yarn (see Table of Materials) and a plastic plate.
      NOTE: Pre-cooling prevents protein degradation. Buffer volumes can be adjusted based on the experimental scale. Ensure all leaves are immersed.
   4. Place the beaker into a vacuum pump. Apply 0.8 MPa vacuum for 1 min, then quickly restore to atmospheric pressure.
   5. Remove leaves from the buffer and gently blot dry with absorbent paper. Roll up leaves, wrap with mesh yarn, and place 5 g of leaves into each 50 mL centrifuge tube. To collect AWF, position rolled leaves tip-side up in tubes with mesh yarn fastened by the tube caps.
   6. Centrifuge at 4 °C, 500 x g for 5 min. Remove leaves and collect the AWF at the bottom of the tube using a pipette. About 5 mL of AWF can be obtained from each tube.
      NOTE: In order to minimize interference from intracellular proteins and to extract the maximum amount of AWF proteins, the extraction can be repeated 2x-3x, but not more than 3x.
2. Total soluble protein extraction
   1. Grind 20 g leaves into fine powder in liquid nitrogen. Add extraction buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 0.034% β-mercaptoethanol (see Table of Materials)) at a 1: 2 ratio.
   2. Shake homogenate in a vertical mixer for 30 min at 4 °C. Centrifuge homogenate at 13,000 x g for 15 min at 4 °C.
   3. Transfer the supernatant to a new tube and repeat centrifugation for 15 min. Transfer the cleared supernatant to another tube.
   4. Filter the supernatant through a 0.22 µm filter (see Table of Materials) to obtain total soluble protein extract.

  

6. Purification of GFP-His with nickel (Ni) affinity chromatography

NOTE: Perform all steps at 4 °C.

1. Add Ni sepharose excel resin (see Table of Materials) to a tube in a 1:1000 ratio relative to the protein extract volume from step 5.1.6. Resin volume equals 1/1000^th of the protein extract volume.
2. Wash Ni resin 3x with 10x resin volume of ddH₂O and PB (pH 7.0) separately in turn and remove the supernatant.
3. Incubate protein extract with Ni resin for 2 h to allow full binding. Centrifuge at 500 x g for 5 min to remove unbound proteins in the supernatant. Wash 3x with 20x resin volume of PB. Centrifuge at 500 x g for 5 min and remove the supernatant.
4. Elute GFP-His by adding 10x resin volume of 250 mM imidazole (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 10% glycerol, 250 mM imidazole) and collect the supernatant.

7. Coomassie blue staining of proteins

1. Add 10 µL of 5x SDS loading buffer to 40 µL of protein samples, boil for 10 min, then run on 15% SDS-PAGE at 110 V for 60 min.
2. Stain protein gels with Coomassie blue solution (0.1% R-250, 25% isopropanol, 10% Glacial acetic acid) for 2 h.
3. Pour off the solution and wash with decolorizing solution for 2 h. Observe the gel; proteins on the gel will be colored blue.

8. Western blotting of proteins

1. Add 10 µL of 5x SDS loading buffer to 40 µL of protein samples, boil for 10 min, then run on 15% SDS-PAGE at 110 V for 60 min.
2. Transfer proteins to a 0.45 µm nitrocellulose membrane at 280 mA. Block the membrane with 5% skimmed milk for 1 h.
3. Dilute the anti-His-tag antibodies at 1:5000 and incubate the membrane with the antibodies for 1 h. Use the tris-buffered saline and tween 20 (TBST) to wash the membrane 4x for 5 min.
4. Dilute the anti-mouse antibodies at 1:10000 and incubate the membrane with the antibodies for 1 h. Use TBST to wash the membrane 4x for 5 min.
5. Mix the developer (see Table of Materials) in the ratio of 1:1. Add 400 µL to the membrane and allow to react for 1 min. Take photographs after 1 min exposure with a visualizer.

9. Protein concentration assay

1. Add 5 µL of protein to 1 mL of Bradford 1x dye reagent and react for 5 min.
2. Determine the OD₅₉₅ value after zeroing and calculate the protein concentration from the standard curve.

10. Protein quantification ³¹

1. Perform Western blotting of protein samples together with standards.
2. Perform quantification of GFP by ratio to standards using ImageJ 1.5.0 greyscale analysis.

  

Representative Results

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 protocols^24,25,26,27, 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.

Discussion

Using plants to produce therapeutic proteins has expanded quickly in recent years^1,2,3,4,5,6. 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 purity^18,19. 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 biology^24,25,26,27, 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.

Materials

Name	Company	Catalog Number	Comments
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