Production of IgG Fusion Proteins Transiently Expressed in Nicotiana benthamiana

Summary

We describe here a simple method for expression, extraction, and purification of recombinant human IgG fused to GFP in Nicotiana benthamiana. This protocol can be extended to purification and visualization of numerous proteins that utilize column chromatography. Moreover, the protocol is adaptable to the in-person and virtual college teaching laboratory, providing project-based exploration.

Abstract

High demand for antibodies as therapeutic interventions for various infectious, metabolic, autoimmune, neoplastic, and other diseases creates a growing need in developing efficient methods for recombinant antibody production. As of 2019, there were more than 70 FDA-approved monoclonal antibodies, and there is exponential growth potential. Despite their promise, limiting factors for widespread use are manufacturing costs and complexity. Potentially, plants offer low-cost, safe, and easily scalable protein manufacturing strategies. Plants like Nicotiana benthamiana not only can correctly fold and assemble complex mammalian proteins but also can add critical post-translational modifications similar to those offered by mammalian cell cultures. In this work, by using native GFP and an acid-stable variant of green fluorescent protein (GFP) fused to human monoclonal antibodies, we were able to visualize the entire transient antibody expression and purification process from N. benthamiana plants. Depending on the experiment's purpose, native GFP fusion can ensure easier visualization during the expression phase in the plants, while acid-stable GFP fusion allows for visualization during downstream processing. This scalable and straightforward procedure can be performed by a single researcher to produce milligram quantities of highly pure antibody or antibody fusion proteins in a matter of days using only a few small plants. Such a technique can be extended to the visualization of any type of antibody purification process and potentially many other proteins, both in plant and other expression systems. Moreover, these techniques can benefit virtual instructions and be executed in a teaching laboratory by undergraduate students possessing minimal prior experience with molecular biology techniques, providing a foundation for project-based exploration with real-world applications.

Introduction

Industry reports indicate that thirteen out of the twenty most-highly grossing drugs in the United States were biologics (protein-based pharmaceuticals), of which nine were antibodies. As of 2019, there were over 570 antibody (Ab) therapeutics at various clinical development phases^1,2,3. Current global Ab sales exceed 100 billion USD, and the monoclonal Ab (mAb) therapeutic market is expected to generate up to 300 billion USD by 2025^1,4. With such high demand and projected increases in revenue, researchers have been working to develop ways to produce Ab therapeutics on an ever-larger scale, with higher quality and lower-costs. Plant-based expression systems have several advantages over traditional mammalian cell lines for the affordable and large-scale manufacture of Ab therapeutics^5,6. Production of protein therapeutics in plants ("molecular pharming") does not require expensive bioreactors or cell culture facilities as do traditional mammalian cell culture techniques^7,8. Plants cannot contract human pathogens, minimizing potential contamination⁹. Both transient and transgenic plant-based protein expression can be utilized as lower-cost alternatives to mammalian or bacterial production systems¹⁰. Though transgenic plants are preferred for crop production, recombinant protein production using this method can require weeks to months. Advances in transient expression using viral vectors through either syringe or vacuum agroinfiltration allow for small- and large-scale production, respectively, of the desired protein in days^11,12,13,14. Production of mAbs against Ebola, Dengue and, Zika, and numerous other recombinant proteins, have been produced and purified quickly and efficiently using transient expression in N. benthamiana plants^{15,16,17,18,19}. These circumstances make transient plant-based expression an attractive option for developing multiple Ab therapeutics and the methods demonstrated in this protocol²⁰.

First-generation mAbs were of murine derivation, which resulted in non-specific immunogenicity when used in human trials²¹. Over time, chimeric, humanized, and eventually, fully human Abs were produced to lessen immunogenicity induced by Ab therapeutics. Unfortunately, some of these Abs still cause host immunogenicity due to differences in glycosylation²¹. Developments in plant engineering have allowed for the modification of Ab glycans, which is essential since an Ab's stability and function can significantly be affected by its glycosylation state²². Advances have allowed production in plant systems of high-level expression of humanized mAbs, containing human glycans and resultantly the desired biological traits of a mass-produced human pharmaceutical^19,21.

In addition to recombinant Abs, Ab fusion molecules (Ab fusions) have been explored for various purposes in recent decades. Ab fusions often consist of an Ab or Ab fragment fused to a molecule or protein and are designed to elicit responses from immune effector cells²³. These molecules have been created as potential therapeutic interventions to treat various pathologies such as cancer and autoimmune diseases^24,25,26,27. Recombinant immune complexes (RICs) are another class of Ab fusions that have been employed as vaccine candidates²⁸. RICs take advantage of the immune system's ability to recognize Fc regions of Ab fusions and have been found to improve immunogenicity when combined with other vaccine platforms^29,30,31. 

Green Fluorescent Protein (GFP) is a bioluminescent protein derived from the jellyfish Aequorea Victoria, which emits green light when excited by ultraviolet light^32,33. Over the years, GFP's use as a visual marker of gene expression has expanded from expression in Escherichia coli to numerous protein expression systems, including N. benthamiana plants^{34,35,36,37,38}. Visible markers, such as GFP, have abundant implications in the teaching and learning of scientific concepts. Numerous entry-level students describe difficulties grasping scientific concepts when the idea being taught is not visible to the naked eye, such as the concepts of molecular biology and related fields³⁹. Visual markers, like GFP, can thus contribute to the processing of information related to the scientific processes and could help lessen the difficulties students report in learning numerous scientific concepts.

Although GFP is often used as a marker to indicate gene and expression in vivo, it is difficult to visualize it in the downstream processes if using acidic conditions. This circumstance is primarily because GFP does not maintain its structure and resultant fluorescence at a low pH⁴⁰. Temporary acidic environments are often required in various purification processes, such as protein G, protein A, and protein L chromatography, often utilized for Ab purification^41,42,43,44. GFP mutants have been used to retain fluorescence under acidic conditions^45,46.

Herein we describe a simple method for expression, extraction, and purification of recombinant IgG fusion proteins in N. benthamiana plants. We produced traditional GFP fused to the N-terminus of a humanized IgG heavy chain, creating a GFP-IgG fusion. Simultaneously, we developed the fusion of a plant codon-optimized sequence for an acid-stable GFP (asGFP) to the N-terminus of a humanized IgG heavy chain, creating an asGFP-IgG fusion. The advantages of producing GFP-IgG include the ability to visualize the presence of a target protein during expression, while asGFP-IgG allows seeing the presence of recombinant protein in not only the expression and extraction steps but also in the purification steps of the protein. This protocol can be adapted for the production, purification, and visualization of a range of GFP fusion proteins produced in N. benthamiana and purified using chromatography techniques that require low pH. The process can also be tailored to various amounts of leaf material. While Abs and fusion proteins tagged with GFP or asGFP are not intended to be used for therapies, these methods can be useful as controls during experiments and can also be further utilized as a teaching tool for molecular and cellular biology and biotechnology, both in-person and virtually.

Protocol

1. Cultivate N. benthamiana plants

1. Place soil peat pellets on a tray and pour previously boiled, still hot (~40-45 °C), water over the peat pellets for full expansion. After pellets are fully expanded, place 2-3 N. benthamiana seeds on each peat pellet using tweezers.
2. Pour about 0.5 in of water to cover the bottom of the tray. Label the tray with the seeding date. Continue to water the seedlings daily with appropriate amounts of fertilizer. Fertilizer (water-soluble all-purpose plant food) concentration is generally 2.5-2.8 g/L.
3. Cover the tray with a humidome top when placed in the growth chamber. Keep the seeded peat pellets in the growth chamber at 23-25 °C, with a 16 h photoperiod and 60% relative humidity.
4. After one week, remove extra plants leaving each pellet with only one seedling.
5. When the plants are 2-3 weeks old, transfer each peat pellet to an individual pot containing moisture control soil. This demonstration used Miracle-Gro moisture control potting soil.
6. Water daily with 1 g/L fertilizer. Never leave the soil completely dry. Plants are ready for infiltration when they are 5-6 weeks old.

2. Preparation of Agrobacterium tumefaciens for infiltration

NOTE: GFP-IgG fusion constructs can be obtained as described in this paper³¹. The asGFP gene was obtained and plant-optimized from this study⁴⁵. The following steps must be done next to a Bunsen burner, and basic aseptic techniques should be applied to avoid contamination.

1. Streak A. tumefaciens EHA105 harboring bean yellow dwarf virus (BeYDV)¹⁹ plant expression vector for each construct (asGFP-IgG, GFP-IgG, light chain) from a glycerol stock on LB agar (10 g/L Tryptone, 10 g/L NaCl, 5 g yeast extract, 15 g/L agar, 50 µg/mL kanamycin) plate. 
2. Grow for one day in a 30 °C standing incubator. Isolate a single colony for verification by standard colony screen PCR protocol. 
3. Use verified colony for each construct. Fill conical tube with 10 mL of LB media (10 g/L tryptone, 10 g/L NaCl, 5 g of yeast extract, 50 µg/mL). Next, add 10 µL of 100 µg/mL kanamycin. Add 10 µL of 2.5 µg/mL rifampicin to prevent E. coli contamination. Incubate at 30°C and 120-150 rpm overnight.
4. The next day, if the Agrobacterium culture is grown to OD₆₀₀ = 0.6-0.9, it can be used for infiltration. If it is overgrown (OD₆₀₀ > 1), 1-2 mL should be transferred to fresh LB with antibiotics and grown to the required OD₆₀₀. Depending on the initial culture's concentration, it may potentially take two days to grow to OD₆₀₀ = 0.6-0.9.
5. Once at appropriate OD₆₀₀, place the cultures in a centrifuge, and pellet the bacteria by centrifugation at 4,500 x g for 20 min, room temperature (RT).
6. Decant supernatant from both samples, and then resuspend each pellet in 1x infiltration buffer (10 mM 2-(N-morpholino) ethanesulfonic acid, 10 mM magnesium sulfate, adjusted to pH 5.5 with KOH) to get final OD₆₀₀ = 0.4. This should take approximately 15-45 mL of infiltration buffer, depending on the initial culture density. Combine equal volumes of each IgG fusion construct with the light chain construct to get final OD₆₀₀ = 0.2 per construct in each tube.

3. Needle-less syringe agroinfiltration

1. Take a straightened paper clip and 5-6-week-old N. benthamiana plants from step 1. Using the paper clip's sharp edge, make a small puncture in the first epidermal layer of the leaf on the adaxial surface. Avoid puncturing it all the way through. 
   NOTE: The lower leaves are easier for infiltration, whereas the leaves on the top of the plant are harder. Generally, the expression of recombinant proteins is highest in the leaves located in the middle of a plant, and these leaves also get less necrotic.
2. Fill a 1 mL syringe, without a needle attached, with the prepared Agrobacterium solution from step 2. Cover the hole made in the previous step with the end of the syringe and slowly push to inject the bacteria into the leaf while applying gentle counterpressure from behind the leaf. Watch the leaf darken as the solution is injected without applying too much pressure on the syringe.
3. Try to infiltrate most of the leaf area at a maximum of 3-4 times – excessive leaf damage may hinder protein yield. The infiltrated plant leaf will appear mostly dark from the bottom view.
   NOTE: This bacterial solution should be enough for at least 3-4 plants per construct. Autoclave any remaining bacterial solution before discarding.

4. Grow and observe the infiltrated N. benthamiana

1. Place infiltrated plants back in the growth chamber and continue to water daily.
2. Observe the leaves for chlorosis and necrosis in infiltrated areas. Observe plants for GFP fluorescence (if GFP is present) under a long and short-wave UV lamp.
3. Day 4-5 shows the highest fluorescence of both GFP constructs in the leaves. Harvest all the leaves at 4-5 dpi (days post-infiltration) and weigh the total leaf material. 
4. Use it immediately for downstream processing or store at -80 °C until ready to use.

5. Protein extraction

1. Keep buffers and blender cups on ice or at 4 °C before use.
2. Prepare 2-3 mL of ice-cold extraction buffer (100 mM Tris-HCl, 50 mM NaCl, 2 mM EDTA, pH 8 with HCl) per 1 g of plant material. Add 2 mM phenylmethylsulfonyl fluoride (PMSF) from stock (100 mM) and 50 mM sodium ascorbate to the extraction buffer just before extraction.
3. Place plant tissue from step 4 into the prechilled blender cup. Add a measured amount of chilled extraction buffer to the blender cup (as indicated in step 5.2). Place the blender cup on the blender. Take a pre-cut sheet of parafilm and stretch it over the top of the blender cup. Blend to homogeneity with 20-sec intervals, stirring well between blend cycles as needed.
4. Transfer blended material to a beaker. Add a stir bar and stir at 4 °C for 30 min to enhance protein solubility and to allow precipitation of solids.
5. Place 2 layers of Miracloth over a clean beaker on ice and pour the extract through it to remove large leaf debris. After all the extract is poured, fold the Miracloth to squeeze the residual leaf extract. The extract should appear dark green without visible particulates.
6. Transfer 50 µL of this sample to a new 1.5 mL tube and label "total extract" for later analysis. Transfer the extract to centrifuge tubes. Centrifuge the remainder of plant extract at 16,000 x g for 20 min, 4 °C and transfer the supernatant to a conical tube.
7. Filter the soluble extract using a 50 mL syringe and syringe glass fiber filter (0.75 µm).
8. Collect 50 µL of a sample after centrifugation, label "soluble extract" for later analysis.

6. Protein G column chromatography procedure

NOTE: The protocol described here is for gravity-flow chromatography using Pierce Protein G agarose resin. If using a different resin, refer to the manufacturer's instructions for adjustments. Never let the resin run dry and prevent all liquid from draining out. Recap the outlet as needed.

1. Set up a polypropylene column that holds 20 mL of sample. 
2. Estimate the amount of slurry needed depending on the target immunoglobulin type and its affinity to the resin. Generally, 3 mL of total slurry with 1.5 mL bed volume is sufficient for the purification of several milligrams of Ab.
3. Carefully pour the required amount of resuspended slurry into the capped column. Open the column outlet from the bottom of the column and allow it to drain until most of the buffer is gone. 
4. Immediately pour 10 mL of wash buffer 1x PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.4 with HCl) on top. Let it drain and repeat this wash step 2x. 
5. Apply the filtered sample from step 5 to the column and collect the flowthrough—aliquot 50 µL of flowthrough for later analysis. Save the rest of the flowthrough in case the Ab did not bind to the resin.
   NOTE: Re-applying flowthrough to a new column does not usually result in a good yield; hence it is advised to start with new leaf material.
6. Wash the resin twice with 10 mL of 1x PBS to reduce non-specific binding. If desired, aliquot 50 µL of wash as the buffer drains through the column to verify that the target Ab is not eluted with a wash buffer. 
7. Set up and label five tubes with 125 µL of sterile 1 M Tris-HCl at pH 8. This is to neutralize the Abs in the acidic elution buffer to avoid potential structural changes. Alternatively, add 30 µL of 2 M Tris base to get a less diluted sample. 
   CAUTION: During elution, UV light may be used for visualization. This does not need to be done for the duration of the elution. If UV is being used, be sure to wear appropriate PPE to avoid damage to eyes and skin. A UV light does not need to be used during the elution step.
8. Elute the Abs by applying 5 mL of elution buffer (100 mM glycine, pH 2.5 with HCl) to the column and collect 1 mL fractions to each designated tube from the previous step. 
9. Immediately regenerate the column by applying 20 mL of wash buffer, followed by 10 mL of wash buffer. Ensure that the resin is not left in an acidic environment for an extended time. Elutions should appear fluorescent, often the highest fluorescence is seen in the second elution but can vary from extraction to extraction.
10. For storage, wash the resin with 10 mL of 20% ethanol in PBS and let it drain halfway. Recap the top, then the bottom of the column, and keep upright at 4 °C. 
    NOTE: Generally, protein G resins can be reused up to 10 times without significant loss of efficiency. Refer to the manufacturer's guidelines for specific details.
11. Determine Ab concentration using a spectrophotometer by measuring absorbance at 280 nm, using the elution buffer as a blank. Store the eluates in -80 °C and aliquot 50 µL of each fraction to a separate tube for further analysis. 

7. SDS-PAGE for GFP-Ig fusion detection

1. Prepare all samples before setting up the SDS-PAGE.
   1. Add 4 µL of sample buffer (6x reducing sample buffer: 3.0 mL of glycerol, 0.93 g of DTT, 1 g of SDS, 7 mL of 4x Tris (pH 6.8) 0.5 M, 1.2 mg of bromophenol blue); (6x non-reducing sample buffer: 3.0 mL of glycerol, 1 g of SDS, 7 mL of 4x Tris (pH 6.8) 0.5 M, 1.2 mg of bromophenol blue) to 20 µL of each sample (total extract, soluble extract, flowthrough, wash, all elution fractions) for analysis. Ensure that tube caps are securely fastened.
   2. Treat only reducing samples for 5 min in a boiling water bath, and then put samples for 5 min on ice. Spin samples in a microcentrifuge for ~5 s and load 20 µL of each sample in the order of collection into the gel wells. Load 3 µL of dual-color protein ladder in a separate well.
2. Run the SDS-PAGE gel at a constant 100 V to desired protein band separation; it takes about 1.5 hours. Monitor the ladder as an indicator of protein separation.
3. Visualize the gel under the UV to observe GFP fluorescence.
4. If desired, stain the gel with Coomassie stain to assess total protein in each sample. Alternatively, perform western blot to evaluate target protein using specific Abs. 
   NOTE: Both Coomassie staining and western blot can be performed by following standard protocols^47,48.

Results

This study demonstrates an easy and fast method to produce recombinant proteins and visualize them throughout downstream processes. Using N. benthamiana and following the provided protocol, recombinant protein production described here can be achieved in less than a week. The overall workflow of plant expression, extraction, and purification is shown in Figure 1. The stages of plant growth from 2-week old seedlings, 4-week old plants, and 6-week old plants are displayed in

Discussion

This protocol can be utilized for the visual verification of any recombinant Ab or recombinant protein produced in N. benthamiana plants, including those that require temporary exposure to acidic environments for column purification purposes^42,43,44. Furthermore, the fusion of asGFP to other proteins in different expression systems can be a useful tool for experimental visualization and education. The protocol herein ca...

Materials

Name	Company	Catalog Number	Comments
5 mL syringe	any	N/A
50 mL syringe	any	N/A
Agar	SIGMA-ALDRICH	A5306
Blender with cups	any	N/A
Bromophenol blue	Bio-Rad	1610404
DTT (DL-Dithiothreitol)	MP BIOMEDICALS	219482101
EDTA (Ethylenedinitrilo)tetraacetic acid	SIGMA-ALDRICH	E-6760
Ethanol	any	N/A
Glycerol	G-Biosciences	BTNM-0037
Glycine	SIGMA-ALDRICH	G7126-500G
HCl (Hydrochloric acid)	EMD MILLIPORE CORPORATION	HX0603-4
Heating block	any reputable supplier	N/A
Jiffy-7 727 w/hole peat pellets	Hummert International	14237000
Kanamycin	Gold Biotechnology Inc	K-120-100
KCl (Potassium Chloride)	SIGMA-ALDRICH	P9541-500G
KH2PO4 (Potassium Phosphate)	J.t.baker	3248-05
KOH (Potassium Hydroxide)	VWR	BDH0262
Magnesium sulfate heptahydrate	SIGMA-ALDRICH	M2773
MES (2-(N-Morpholino)ethanesulfonic acid)	SIGMA-ALDRICH	M8250
Miracloth	Millipore	4 75855-1R
Moisture control potting mix	Miracle-Gro	755783
Na2HPO4 (Sodium Phosphate)	J.t.baker	3827-01
NaCl (Sodium Chloride)	Santa Cruz Biotechnology	sc-203274C
Nicotiana benthamiana seeds	any reputable supplier	N/A
PMSF (Phenylmethylsulfonyl Fluoride)	G-Biosciences	786-787
Polypropylene Column	any	N/A
Precision Plus Protein Dual Color Standards	Bio-Rad	1610394
Protein G resin	Thermo Fisher Scientific	20399
Rifampicin	Gold Biotechnology Inc	R-120-25
SDS (Sodium Dodecyl Sulfate)	G-Biosciences	DG093
Sodium Ascorbate	SIGMA-ALDRICH	A7631-500G
Spectrophotometer	any reputable supplier	N/A
Titan3 0.75 µm glass fiber filter	ThermoScientific	40725-GM
Tray for peat pellets with dome	any	N/A
TRIS Base	J.t.baker	4109-02
Tris-HCl	Amresco	M108-1KG
Tryptone	SIGMA-ALDRICH	17221
UV lamp	any	N/A
Water Soluble All Purpose Plant Food	Miracle-Gro	2000992
Yeast extract	SIGMA-ALDRICH	9182