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Method Article

Production of IgG Fusion Proteins Transiently Expressed in Nicotiana benthamiana

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DOI:

10.3791/61774

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January 16th, 2021

 ,  ,  ,  ,  ,  , 
1Center for Immunotherapy, Vaccines and Virotherapy, The Biodesign Institute, Arizona State University, 2School of Life Sciences, Arizona State University, 3Molecular Biosciences/Biotechnology Undergraduate Program, Arizona State University, 4Department of Microbiology and Immunology, University of Michigan Medical School

* These authors contributed equally

In This Article

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 phases1,2,3. 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 20251,4. 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 therapeutics5,6. Production of protein therapeutics in plants ("molecular pharming") does not require expensive bioreactors or cell culture facilities as do traditional mammalian cell culture techniques7,8. 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 days11,12,13,14. 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 plants15,16,17,18,19. 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 pharmaceutical19,21.

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 diseases24,25,26,27. 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 platforms29,30,31. 

Green Fluorescent Protein (GFP) is a bioluminescent protein derived from the jellyfish Aequorea Victoria, which emits green light when excited by ultraviolet light32,33. 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 plants34,35,36,37,38. 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 purification41,42,43,44. GFP mutants have been used to retain fluorescence under acidic conditions45,46.

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.

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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 protocols47,48.

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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

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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 purposes42,43,44. 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...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

We thank Maria Pia DiPalma for editing the video. We also thank the Office of Educational Outreach and Student Services at Arizona State University for their generous publication fee assistance. Research for this protocol was supported by the School of Life Sciences, Arizona State University.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
5 mL syringeanyN/A
50 mL syringeanyN/A
AgarSIGMA-ALDRICHA5306
Blender with cupsanyN/A
Bromophenol blueBio-Rad1610404
DTT (DL-Dithiothreitol)MP BIOMEDICALS219482101
EDTA (Ethylenedinitrilo)tetraacetic acidSIGMA-ALDRICHE-6760
EthanolanyN/A
GlycerolG-BiosciencesBTNM-0037
GlycineSIGMA-ALDRICHG7126-500G
HCl (Hydrochloric acid)EMD MILLIPORE CORPORATIONHX0603-4
Heating blockany reputable supplierN/A
Jiffy-7 727 w/hole peat pelletsHummert International14237000
KanamycinGold Biotechnology IncK-120-100
KCl (Potassium Chloride)SIGMA-ALDRICHP9541-500G
KH2PO4 (Potassium Phosphate)J.t.baker3248-05
KOH (Potassium Hydroxide)VWRBDH0262
Magnesium sulfate heptahydrateSIGMA-ALDRICHM2773
MES (2-(N-Morpholino)ethanesulfonic acid)SIGMA-ALDRICHM8250
MiraclothMillipore4 75855-1R
Moisture control potting mixMiracle-Gro755783
Na2HPO4 (Sodium Phosphate)J.t.baker3827-01
NaCl (Sodium Chloride)Santa Cruz Biotechnologysc-203274C
Nicotiana benthamiana seedsany reputable supplierN/A
PMSF (Phenylmethylsulfonyl Fluoride)G-Biosciences786-787
Polypropylene ColumnanyN/A
Precision Plus Protein Dual Color StandardsBio-Rad1610394
Protein G resinThermo Fisher Scientific20399
RifampicinGold Biotechnology IncR-120-25
SDS (Sodium Dodecyl Sulfate)G-BiosciencesDG093
Sodium AscorbateSIGMA-ALDRICHA7631-500G
Spectrophotometerany reputable supplierN/A
Titan3 0.75 µm glass fiber filterThermoScientific40725-GM
Tray for peat pellets with domeanyN/A
TRIS BaseJ.t.baker4109-02
Tris-HClAmrescoM108-1KG
TryptoneSIGMA-ALDRICH17221
UV lampanyN/A
Water Soluble All Purpose Plant FoodMiracle-Gro2000992
Yeast extractSIGMA-ALDRICH9182

References

  1. Lu, R. M., et al. Development of therapeutic antibodies for the treatment of diseases. Journal of Biomedical Science. 27 (1), (2020).
  2. Kaplon, H., Reichert, J. M. Antibodies to watch in 2019. mAbs. 11 (2), 219-238 (2019).
  3. The top 20 drugs by U.S. sales. FiercePharma. , Available from: https://www.fiercepharma.com/special-report/top-20-drugs-by-2018-u-s-sales (2018).
  4. Grilo, A. L., Mantalaris, A. The Increasingly Human and Profitable Monoclonal Antibody Market. Trends in Biotechnology. 37 (1), 9-16 (2019).
  5. Kim, M. -Y., et al. Novel vaccination approach for dengue infection based on recombinant immune complex universal platform. Vaccine. 33 (15), 1830-1838 (2015).
  6. Twyman, R. M., Schillberg, S., Fischer, R. Transgenic plants in the biopharmaceutical market. Expert Opinion on Emerging Drugs. 10 (1), 185-218 (2005).
  7. Stoger, E., Fischer, R., Moloney, M., Ma, J. K. -C. Plant Molecular Pharming for the Treatment of Chronic and Infectious Diseases. Annual Review of Plant Biology. 65 (1), 743-768 (2014).
  8. Buyel, J. F., Fischer, R. Predictive models for transient protein expression in tobacco (Nicotiana tabacum L.) can optimize process time, yield, and downstream costs. Biotechnology and Bioengineering. 109 (10), 2575-2588 (2012).
  9. Yao, J., Weng, Y., Dickey, A., Wang, K. Y. Plants as factories for human pharmaceuticals: Applications and challenges. International Journal of Molecular Sciences. 16 (12), 28549-28565 (2015).
  10. Mor, T. S., Moon, Y. -S., Palmer, K. E., Mason, H. S. Geminivirus vectors for high-level expression of foreign proteins in plant cells. Biotechnology and Bioengineering. 81 (4), 430-437 (2003).
  11. Hefferon, K. L. Plant virus expression vectors set the stage as production platforms for biopharmaceutical proteins. Virology. 433 (1), 1-6 (2012).
  12. Yamamoto, T., et al. Improvement of the transient expression system for production of recombinant proteins in plants. Scientific Reports. 8 (1), 4755(2018).
  13. Zhong, G. Y., et al. Commercial production of aprotinin in transgenic maize seeds. Molecular Breeding. 5 (4), 345-356 (1999).
  14. Giddings, G., Allison, G., Brooks, D., Carter, A. Transgenic plants as factories for biopharmaceuticals. Nature Biotechnology. 18 (11), 1151-1155 (2000).
  15. Fulton, A., Lai, H., Chen, Q., Zhang, C. Purification of monoclonal antibody against Ebola GP1 protein expressed in Nicotiana benthamiana. Journal of Chromatography A. 1389, 128-132 (2015).
  16. Diamos, A., et al. A highly expressing, soluble, and stable plant-made IgG fusion carrying Zika virus envelope domain III elicits potent immunogenic responses in mice without adjuvant. Frontiers in immunology. 3140 (11), (2020).
  17. Hunter, J. G. L., et al. Evaluation of a toxoid fusion protein vaccine produced in plants to protect poultry against necrotic enteritis. PeerJ. 2019 (3), 6600(2019).
  18. Dent, M., et al. Plant-produced anti-dengue virus monoclonal antibodies exhibit reduced antibody-dependent enhancement of infection activity. Journal of General Virology. 97 (12), 3280-3290 (2016).
  19. Diamos, A. G., et al. High Level Production of Monoclonal Antibodies Using an Optimized Plant Expression System. Frontiers in Bioengineering and Biotechnology. 7, 472(2020).
  20. Olinger, G. G., et al. Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques. Proceedings of the National Academy of Sciences of the United States of America. 109 (44), 18030-18035 (2012).
  21. Mastrangeli, R., Palinsky, W., Bierau, H. Glycoengineered antibodies: towards the next-generation of immunotherapeutics. Glycobiology. 29 (3), 199-210 (2019).
  22. Montero-Morales, L., Steinkellner, H. Advanced Plant-Based Glycan Engineering. Frontiers in Bioengineering and Biotechnology. 6, 81(2018).
  23. Joosten, V., Lokman, C., vanden Hondel, C. A. M. J. J., Punt, P. J. The production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi. Microbial Cell Factories. 2 (1), 1-15 (2003).
  24. Müller, D. Antibody fusions with immunomodulatory proteins for cancer therapy. Pharmacology and Therapeutics. 154, 57-66 (2015).
  25. Bootz, F., Neri, D. Immunocytokines: A novel class of products for the treatment of chronic inflammation and autoimmune conditions. Drug Discovery Today. 21 (1), 180-189 (2016).
  26. Jafari, R., Zolbanin, N. M., Rafatpanah, H., Majidi, J., Kazemi, T. Fc-fusion Proteins in Therapy: An Updated View. Current Medicinal Chemistry. 24 (12), (2017).
  27. Brinkmann, U., Kontermann, R. E. The making of bispecific antibodies. mAbs. 9 (2), 182-212 (2017).
  28. Mason, H. S. Recombinant immune complexes as versatile and potent vaccines. Human Vaccines & Immunotherapeutics. 12 (4), 988-989 (2016).
  29. Mason, H. S. Recombinant immune complexes as versatile and potent vaccines. Human Vaccines and Immunotherapeutics. 12 (4), 988-989 (2016).
  30. Diamos, A. G., et al. Vaccine synergy with virus-like particle and immune complex platforms for delivery of human papillomavirus L2 antigen. Vaccine. 37 (1), 137-144 (2019).
  31. Diamos, A. G., et al. Codelivery of improved immune complex and virus-like particle vaccines containing Zika virus envelope domain III synergistically enhances immunogenicity. Vaccine. 38 (18), 3455-3463 (2020).
  32. Prasher, D. C., Eckenrode, V. K., Ward, W. W., Prendergast, F. G., Cormier, M. J. Primary structure of the Aequorea victoria green-fluorescent protein. Gene. 111 (2), 229-233 (1992).
  33. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., Prasher, D. C. Green fluorescent protein as a marker for gene expression. Science. 263 (5148), 802-805 (1994).
  34. Palm, M., Baumstark-Khan, C., Horneck, G. Green Fluorescent Protein (GFP) Expression in Mammalian Cells After UV-Irradiation. Fundamentals for the Assessment of Risks from Environmental Radiation. , 311-316 (1999).
  35. Paemeleire, K., et al. Intercellular calcium waves in HeLa cells expressing GFP-labeled connexin 43, 32, or 26. Molecular Biology of the Cell. 11 (5), 1815-1827 (2000).
  36. Harper, B. K., Stewart, C. N. Patterns of Green Fluorescent Protein Expression in Transgenic Plants. Plant Molecular Biology Reporter. 18 (2), 141(2000).
  37. Kaishima, M., Ishii, J., Matsuno, T., Fukuda, N., Kondo, A. Expression of varied GFPs in Saccharomyces cerevisiae: Codon optimization yields stronger than expected expression and fluorescence intensity. Scientific Reports. 6, (2016).
  38. Glow in the Dark: Fluorescent Proteins as Cell and Tissue-Specific Markers in Plants. , Available from: https://pubmed.ncbi.nlm.nih.gov/21772029/ (2020).
  39. Mnguni, L. E. The theoretical cognitive process of visualization for science education. SpringerPlus. 3 (1), 1-9 (2014).
  40. Shaner, N. C., Patterson, G. H., Davidson, M. W. Advances in fluorescent protein technology. Journal of Cell Science. 120 (24), 4247-4260 (2007).
  41. Bjorck, L., Kronvall, G. Purification and some properties of streptococcal protein G, a novel IgG-Binding reagent. The Journal of Immunology. 133 (2), (1984).
  42. Andrew, S. M., Titus, J. A. Purification of Immunoglobulin G. Current Protocols in Immunology. 21 (1), 1-12 (2001).
  43. Hober, S., Nord, K., Linhult, M. Protein A chromatography for antibody purification. Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences. 848 (1), 40-47 (2007).
  44. Vola, R., Lombardi, A., Tarditi, L., Björck, L., Mariani, M. Recombinant proteins L and LG: efficient tools for purification of murine immunoglobulin G fragments. Journal of Chromatography B: Biomedical Sciences and Applications. 668 (2), 209-218 (1995).
  45. Shinoda, H., Ma, Y., Nakashima, R., Sakurai, K., Matsuda, T., Nagai, T. Acid-Tolerant Monomeric GFP from Olindias formosa. Cell Chemical Biology. 25 (3), 330-338 (2018).
  46. Roberts, T. M., et al. Identification and Characterisation of a pH-stable GFP. Scientific Reports. 6 (1), 1-9 (2016).
  47. Brunelle, J. L., Green, R. Coomassie blue staining. Methods in Enzymology. 541, 161-167 (2014).
  48. Mahmood, T., Western Yang, P. C. Western blot: Technique, theory, and trouble shooting. North American Journal of Medical Sciences. 4 (9), 429-434 (2012).
  49. Vessoni Penna, T. C., Ishii, M., Cholewa, O., de Souza, L. C. Thermal characteristics of recombinant green fluorescent protein (GFPuv) extracted from Escherichia coli. Letters in Applied Microbiology. 38 (2), 135-139 (2004).
  50. Kang, S. J., Park, E. A., Lee, D. H., Hong, K. W. Comparison of the stability of eGFP displayed on the Bacillus subtilis spore surface using CotB and C-terminally truncated CotB proteins as an anchoring motif under extreme conditions. Applied Biological Chemistry. 62 (1), 41(2019).
  51. Kuta, D. D., Tripathi, L. Agrobacterium-induced hypersensitive necrotic reaction in plant cells: a resistance response against Agrobacterium-mediated DNA transfer. African Journal of Biotechnology. 4 (8), 752-757 (2005).
  52. Qiusheng, Z., Bao, J., Likun, L., Xianhua, X. Effects of antioxidants on the plant regeneration and GUS expressive frequency of peanut (Arachis hypogaea) explants by Agrobacterium tumefaciens. Plant Cell, Tissue and Organ Culture. 81 (1), 83-90 (2005).
  53. Diamos, A. G., Rosenthal, S. H., Mason, H. S. H.S. 5' and 3' Untranslated Regions Strongly Enhance Performance of Geminiviral Replicons in Nicotiana benthamiana Leaves. Frontiers in Plant Science. 7, 200(2016).
  54. Huang, Z., Chen, Q., Hjelm, B., Arntzen, C., Mason, H. A DNA replicon system for rapid high-level production of virus-like particles in plants. Biotechnology and Bioengineering. 103 (4), 706-714 (2009).
  55. Diamos, A. G., Mason, H. S. Modifying the Replication of Geminiviral Vectors Reduces Cell Death and Enhances Expression of Biopharmaceutical Proteins in Nicotiana benthamiana Leaves. Frontiers in Plant Science. 9, 1974(2019).
  56. Gordon-Kamm, W., et al. Stimulation of the cell cycle and maize transformation by disruption of the plant retinoblastoma pathway. Proceedings of the National Academy of Sciences of the United States of America. 99 (18), 11975-11980 (2002).
  57. Villemont, E., Dubois, F., Sangwan, R. S., Vasseur, G., Bourgeois, Y., Sangwan-Norreel, B. S. Role of the host cell cycle in the Agrobacterium-mediated genetic transformation of Petunia: Evidence of an S-phase control mechanism for T-DNA transfer. Planta. 201 (2), 160-172 (1997).
  58. Regnard, G. L., Halley-Stott, R. P., Tanzer, F. L., Hitzeroth, I. I., Rybicki, E. P. High level protein expression in plants through the use of a novel autonomously replicating geminivirus shuttle vector. Plant Biotechnology Journal. 8 (1), 38-46 (2010).
  59. Chen, Q., He, J., Phoolcharoen, W., Mason, H. S. Geminiviral vectors based on bean yellow dwarf virus for production of vaccine antigens and monoclonal antibodies in plants. Human Vaccines. 7 (3), 331-338 (2011).
  60. Morton, T. G., Gold, A. J., Sullivan, W. M. Influence of Overwatering and Fertilization on Nitrogen Losses from Home Lawns. Journal of Environmental Quality. 17 (1), 124-130 (1988).
  61. Brown, J. C. Iron Chlorosis in Plants. Advances in Agronomy. 13, 329-369 (1961).
  62. Morton, E. R., Fuqua, C. Laboratory Maintenance of Agrobacterium. Current Protocols in Microbiology. , CHAPTER, Unit3D.1 (2012).

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