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.

1. Cultivate N. benthamiana plants
<ol>
	<li>Place soil peat pellets on a tray and pour previously boiled, still hot (~40-45 &#176;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.</li>
	<li>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.</li>
	<li>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 &#176;C, with a 16 h photoperiod and 60% relative humidity.</li>
	<li>After one week, remove extra plants leaving each pellet with only one seedling.</li>
	<li>When the plants are 2-3 weeks old, transfer each peat pellet to an individual pot containing moisture control soil.&#160;This demonstration used Miracle-Gro moisture control potting soil.</li>
	<li>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.</li>
</ol>
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.
<ol>
	<li>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&#160;stock on LB agar (10 g/L Tryptone, 10 g/L NaCl, 5 g yeast extract, 15 g/L agar, 50 &#181;g/mL kanamycin) plate.&#160;</li>
	<li>Grow for one day in a 30 &#176;C standing incubator. Isolate a single colony for verification by standard colony screen PCR protocol.&#160;</li>
	<li>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 &#181;g/mL). Next, add 10 &#181;L of 100 &#181;g/mL kanamycin. Add 10 &#181;L of 2.5 &#181;g/mL rifampicin to prevent E. coli contamination. Incubate at 30&#176;C and 120-150 rpm overnight.</li>
	<li>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 &#62; 1), 1-2 mL should be transferred to fresh LB with antibiotics and grown to the required OD600.&#160;Depending on the initial culture&#39;s concentration, it may potentially take two days to grow to OD600 = 0.6-0.9.</li>
	<li>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).</li>
	<li>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.</li>
</ol>
3. Needle-less syringe agroinfiltration
<ol>
	<li>Take a straightened paper clip and 5-6-week-old N. benthamiana plants from step 1. Using the paper clip&#39;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.&#160; 
	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.</li>
	<li>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.</li>
	<li>Try to infiltrate most of the leaf area at a maximum of 3-4 times &#8211; excessive leaf damage may hinder protein yield.&#160;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.</li>
</ol>
4. Grow and observe the infiltrated N. benthamiana
<ol>
	<li>Place infiltrated plants back in the growth chamber and continue to water daily.</li>
	<li>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.</li>
	<li>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.&#160;</li>
	<li>Use it immediately for downstream processing or store at -80 &#176;C until ready to use.</li>
</ol>
5. Protein extraction
<ol>
	<li>Keep buffers and blender cups on ice or at 4 &#176;C before use.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Transfer blended material to a beaker. Add a stir bar and stir at 4 &#176;C for 30 min to enhance protein solubility and to allow precipitation of solids.</li>
	<li>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.&#160;The extract should appear dark green without visible particulates.</li>
	<li>Transfer 50 &#181;L of this sample to a new 1.5 mL tube and label &#34;total extract&#34; for later analysis. Transfer the extract to centrifuge tubes. Centrifuge the remainder of plant extract at 16,000 x g for 20 min, 4 &#176;C and transfer the supernatant to a conical tube.</li>
	<li>Filter the soluble extract using a 50 mL syringe and syringe glass fiber filter (0.75 &#181;m).</li>
	<li>Collect 50 &#181;L of a sample after centrifugation, label &#34;soluble extract&#34; for later analysis.</li>
</ol>
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&#39;s instructions for adjustments. Never let the resin run dry and prevent all liquid from draining out. Recap the outlet as needed.
<ol>
	<li>Set up a polypropylene column that holds 20 mL of sample.&#160;</li>
	<li>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.</li>
	<li>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.&#160;</li>
	<li>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.&#160;</li>
	<li>Apply the filtered sample from step 5 to the column and collect the flowthrough&#8212;aliquot 50 &#181;L 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.</li>
	<li>Wash the resin twice with 10 mL of 1x PBS to reduce non-specific binding. If desired, aliquot 50 &#181;L of wash as the buffer drains through the column to verify that the target Ab is not eluted with a wash buffer.&#160;</li>
	<li>Set up and label five tubes with 125 &#181;L 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 &#181;L of 2 M Tris base to get a less diluted sample.&#160; 
	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.</li>
	<li>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.&#160;</li>
	<li>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.</li>
	<li>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 &#176;C.&#160; 
	NOTE: Generally, protein G resins can be reused up to 10 times without significant loss of efficiency. Refer to the manufacturer&#39;s guidelines for specific details.</li>
	<li>Determine Ab concentration using a spectrophotometer by measuring absorbance at 280 nm, using the elution buffer as a blank. Store the eluates in -80 &#176;C and aliquot 50 &#181;L of each fraction to a separate tube for further analysis.&#160;</li>
</ol>
7. SDS-PAGE for GFP-Ig fusion detection
<ol>
	<li>Prepare all samples before setting up the SDS-PAGE.
	<ol>
		<li>Add 4 &#181;L 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 &#181;L of each sample (total extract, soluble extract, flowthrough, wash, all elution fractions) for analysis. Ensure that tube caps are securely fastened.</li>
		<li>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 &#181;L of each sample in the order of collection into the gel wells. Load 3 &#181;L of dual-color protein ladder in a separate well.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Visualize the gel under the UV to observe GFP fluorescence.</li>
	<li>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.&#160; 
	NOTE: Both Coomassie staining and western blot can be performed by following standard protocols47,48.</li>
</ol>

The authors have nothing to disclose.

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...

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 (&#34;molecular pharming&#34;) 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&#39;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&#39;s ability to recognize Fc regions of Ab fusions and have been found to improve immunogenicity when combined with other vaccine platforms29,30,31.&#160;
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&#39;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.

<table border="0" cellpadding="0" cellspacing="0" style="width:646px;" width="644">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:262px;">5 mL syringe</td>
			<td style="width:174px;">any</td>
			<td style="width:119px;">N/A</td>
			<td style="width:89px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:262px;">50 mL syringe</td>
			<td style="width:174px;">any</td>
			<td style="width:119px;">N/A</td>
			<td style="width:89px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:262px;">Agar</td>
			<td style="width:174px;">SIGMA-ALDRICH</td>
			<td style="width:119px;">A5306</td>
			<td style="width:89px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:262px;">Blender with cups</td>
			<td style="width:174px;">any</td>
			<td style="width:119px;">N/A</td>
			<td style="width:89px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:262px;">Bromophenol blue</td>
			<td style="width:174px;">Bio-Rad</td>
			<td style="width:119px;">1610404</td>
			<td style="width:89px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:262px;">DTT (DL-Dithiothreitol)</td>
			<td style="width:174px;">MP BIOMEDICALS</td>
			<td style="width:119px;">219482101</td>
			<td style="width:89px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:262px;">EDTA (Ethylenedinitrilo)tetraacetic acid</td>
			<td style="width:174px;">SIGMA-ALDRICH</td>
			<td style="width:119px;">E-6760</td>
			<td style="width:89px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:262px;">Ethanol</td>
			<td style="width:174px;">any</td>
			<td style="width:119px;">N/A</td>
			<td style="width:89px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:262px;">Glycerol</td>
			<td style="width:174px;">G-Biosciences</td>
			<td style="width:119px;">BTNM-0037</td>
			<td style="width:89px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:262px;">Glycine</td>
			<td style="width:174px;">SIGMA-ALDRICH</td>
			<td style="width:119px;">G7126-500G</td>
			<td style="width:89px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:262px;">HCl (Hydrochloric acid)</td>
			<td style="width:174px;">EMD MILLIPORE CORPORATION</td>
			<td style="width:119px;">HX0603-4</td>
			<td style="width:89px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:262px;">Heating block</td>
			<td style="width:174px;">any reputable supplier</td>
			<td style="width:119px;">N/A</td>
			<td style="width:89px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:262px;">Jiffy-7 727 w/hole peat pellets</td>
			<td style="width:174px;">Hummert International</td>
			<td style="width:119px;">14237000</td>
			<td style="width:89px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:262px;">Kanamycin</td>
			<td style="width:174px;">Gold Biotechnology Inc</td>
			<td style="width:119px;">K-120-100</td>
			<td style="width:89px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:262px;">KCl (Potassium Chloride)</td>
			<td style="width:174px;">SIGMA-ALDRICH</td>
			<td style="width:119px;">P9541-500G</td>
			<td style="width:89px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:262px;">KH2PO4 (Potassium Phosphate)</td>
			<td style="width:174px;">J.t.baker</td>
			<td style="width:119px;">3248-05</td>
			<td style="width:89px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:262px;">KOH (Potassium Hydroxide)</td>
			<td style="width:174px;">VWR</td>
			<td style="width:119px;">BDH0262</td>
			<td style="width:89px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:262px;">Magnesium sulfate heptahydrate</td>
			<td style="width:174px;">SIGMA-ALDRICH</td>
			<td style="width:119px;">M2773</td>
			<td style="width:89px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:262px;">MES (2-(N-Morpholino)ethanesulfonic acid)</td>
			<td style="width:174px;">SIGMA-ALDRICH</td>
			<td style="width:119px;">M8250</td>
			<td style="width:89px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:262px;">Miracloth</td>
			<td style="width:174px;">Millipore</td>
			<td style="width:119px;">4 75855-1R</td>
			<td style="width:89px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:262px;">Moisture control potting mix</td>
			<td style="width:174px;">Miracle-Gro</td>
			<td style="width:119px;">755783</td>
			<td style="width:89px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:262px;">Na2HPO4 (Sodium Phosphate)</td>
			<td style="width:174px;">J.t.baker</td>
			<td style="width:119px;">3827-01</td>
			<td style="width:89px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:262px;">NaCl (Sodium Chloride)</td>
			<td style="width:174px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-203274C</td>
			<td style="width:89px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:262px;">Nicotiana benthamiana seeds</td>
			<td style="width:174px;">any reputable supplier</td>
			<td style="width:119px;">N/A</td>
			<td style="width:89px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:262px;">PMSF (Phenylmethylsulfonyl Fluoride)</td>
			<td style="width:174px;">G-Biosciences</td>
			<td style="width:119px;">786-787</td>
			<td style="width:89px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:262px;">Polypropylene Column</td>
			<td style="width:174px;">any</td>
			<td style="width:119px;">N/A</td>
			<td style="width:89px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:262px;">Precision Plus Protein Dual Color Standards</td>
			<td style="width:174px;">Bio-Rad</td>
			<td style="width:119px;">1610394</td>
			<td style="width:89px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:262px;">Protein G resin</td>
			<td style="width:174px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">20399</td>
			<td style="width:89px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:262px;">Rifampicin</td>
			<td style="width:174px;">Gold Biotechnology Inc</td>
			<td style="width:119px;">R-120-25</td>
			<td style="width:89px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:262px;">SDS (Sodium Dodecyl Sulfate)</td>
			<td style="width:174px;">G-Biosciences</td>
			<td style="width:119px;">DG093</td>
			<td style="width:89px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:262px;">Sodium Ascorbate</td>
			<td style="width:174px;">SIGMA-ALDRICH</td>
			<td style="width:119px;">A7631-500G</td>
			<td style="width:89px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:262px;">Spectrophotometer</td>
			<td style="width:174px;">any reputable supplier</td>
			<td style="width:119px;">N/A</td>
			<td style="width:89px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:262px;">Titan3 0.75 &#181;m glass fiber filter</td>
			<td style="width:174px;">ThermoScientific</td>
			<td style="width:119px;">40725-GM</td>
			<td style="width:89px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:262px;">Tray for peat pellets with dome</td>
			<td style="width:174px;">any</td>
			<td style="width:119px;">N/A</td>
			<td style="width:89px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:262px;">TRIS Base</td>
			<td style="width:174px;">J.t.baker</td>
			<td style="width:119px;">4109-02</td>
			<td style="width:89px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:262px;">Tris-HCl</td>
			<td style="width:174px;">Amresco</td>
			<td style="width:119px;">M108-1KG</td>
			<td style="width:89px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:262px;">Tryptone</td>
			<td style="width:174px;">SIGMA-ALDRICH</td>
			<td style="width:119px;">17221</td>
			<td style="width:89px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:262px;">UV lamp</td>
			<td style="width:174px;">any</td>
			<td style="width:119px;">N/A</td>
			<td style="width:89px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:262px;">Water Soluble All Purpose Plant Food</td>
			<td style="width:174px;">Miracle-Gro</td>
			<td style="width:119px;">2000992</td>
			<td style="width:89px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:262px;">Yeast extract</td>
			<td style="width:174px;">SIGMA-ALDRICH</td>
			<td style="width:119px;">9182</td>
			<td style="width:89px;"></td>
		</tr>
	</tbody>
</table>

production of igg fusion proteins transiently expressed in nicotiana benthamiana

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&#160;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&#39;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.

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 <strong c...

Watch this Scientific Journal Video about Production of IgG Fusion Proteins Transiently Expressed in Nicotiana benthamiana at JoVE.com

Production of IgG Fusion Proteins Transiently Expressed in Nicotiana benthamiana

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.

Production of IgG Fusion Proteins Transiently Expressed in Nicotiana benthamiana

High demand for antibodies as therapeutic interventions for various infectious, metabolic, autoimmune, neoplastic, and other diseases creates a growing ...

production-igg-fusion-proteins-transiently-expressed-nicotiana

Research

JoVE Journal

Biology

7.2K Views.  Arizona State University. 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.

Video: Production of IgG Fusion Proteins Transiently Expressed in Nicotiana benthamiana

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5.
Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2.
Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. These systems offer a broad selection of fluorescent substrates optimized for a range of imaging instrumentation. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again. There are two steps to using this system: cloning and expression of the protein of interest as a SNAP-tag fusion, and labeling of the fusion with the SNAP-tag substrate of choice. The SNAP-tag is a small protein based on human O6-alkylguanine-DNA-alkyltransferase (hAGT), a DNA repair protein. SNAP-tag labels are dyes conjugated to guanine or chloropyrimidine leaving groups via a benzyl linker. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag. CLIP-tag is a modified version of SNAP-tag, engineered to react with benzylcytosine rather than benzylguanine derivatives. When used in conjunction with SNAP-tag, CLIP-tag enables the orthogonal and complementary labeling of two proteins simultaneously in the same cells.

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again.

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Analysis of SNARE-mediated Membrane Fusion Using an Enzymatic Cell Fusion Assay

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and on target membranes (t-SNAREs) catalyze intracellular vesicle fusion1-4. Reconstitution assays are essential for dissecting the mechanism and regulation of SNARE-mediated membrane fusion5. In a cell fusion assay6,7, SNARE proteins are expressed ectopically at the cell surface. These "flipped" SNARE proteins drive cell-cell fusion, demonstrating that SNAREs are sufficient to fuse cellular membranes. Because the cell fusion assay is based on microscopic analysis, it is less efficient when used to analyze multiple v- and t-SNARE interactions quantitatively.
Here we describe a new assay8 that quantifies SNARE-mediated cell fusion events by activated expression of &beta;-galactosidase. Two components of the Tet-Off gene expression system9 are used as a readout system: the tetracycline-controlled transactivator (tTA) and a reporter plasmid that encodes the LacZ gene under control of the tetracycline-response element (TRE-LacZ). We transfect tTA into COS-7 cells that express flipped v-SNARE proteins at the cell surface (v-cells) and transfect TRE-LacZ into COS-7 cells that express flipped t-SNARE proteins at the cell surface (t-cells). SNARE-dependent fusion of the v- and t-cells results in the binding of tTA to TRE, the transcriptional activation of LacZ and expression of &beta;-galactosidase. The activity of &beta;-galactosidase is quantified using a colorimetric method by absorbance at 420 nm.
The vesicle-associated membrane proteins (VAMPs) are v-SNAREs that reside in various post-Golgi vesicular compartments10-15. By expressing VAMPs 1, 3, 4, 5, 7 and 8 at the same level, we compare their membrane fusion activities using the enzymatic cell fusion assay. Based on spectrometric measurement, this assay offers a quantitative approach for analyzing SNARE-mediated membrane fusion and for high-throughput studies.

We have developed a cell fusion assay that quantifies SNARE-mediated membrane fusion events by activated expression of &beta;-galactosidase.

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)  proteins on vesicles (v-SNAREs) and on target membranes ...

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most commonly used agent for agroinfiltration is Agrobacterium tumefaciens, a pathogen of many dicot plant species. This implies that agroinfiltration can be applied to many plant species. Here, we present our protocols and expected results when applying these methods to the potato (Solanum tuberosum), its related wild tuber-bearing Solanum species (Solanum section Petota) and the model plant Nicotiana benthamiana. In addition to functional analysis of single genes, such as resistance (R) or avirulence (Avr) genes, the agroinfiltration assay is very suitable for recapitulating the R-AVR interactions associated with specific host pathogen interactions by simply delivering R and Avr transgenes into the same cell. However, some plant genotypes can raise nonspecific defense responses to Agrobacterium, as we observed for example for several potato genotypes. Compared to agroinfiltration, detection of AVR activity with PVX agroinfection is more sensitive, more high-throughput in functional screens and less sensitive to nonspecific defense responses to Agrobacterium. However, nonspecific defense to PVX can occur and there is a risk to miss responses due to virus-induced extreme resistance. Despite such limitations, in our experience, agroinfiltration and PVX agroinfection are both suitable and complementary assays that can be used simultaneously to confirm each other&#39;s results.

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are routine functional assays for transient ectopic expression of genes in plants. These methods are efficient assays in effectoromics strategies (rapid resistance and avirulence gene discovery) and crucial to modern research in molecular plant pathology. They meet the demand for robust high-throughput functional analysis in plants.

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most ...

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana &#215; N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel protein-protein interaction pairs and monitoring protein interaction dynamics are of particular interest for revealing how plants respond to environmental factors and/or developmental signals. A plethora of approaches have been developed to examine protein-protein interactions, either in vitro or in vivo. Among them, the recently established luciferase complementation imaging (LCI) assay is the simplest and fastest method for demonstrating in vivo protein-protein interactions. In this assay, protein A or protein B is fused with the amino-terminal or carboxyl-terminal half of luciferase, respectively. When protein A interacts with protein B, the two halves of luciferase will be reconstituted to form a functional and active luciferase enzyme. Luciferase activity can be recorded with a luminometer or CCD-camera. Compared with other approaches, the LCI assay shows protein-protein interactions both qualitatively and quantitatively. Agrobacterium infiltration in Nicotiana benthamiana leaves is a widely used system for transient protein expression. With the combination of LCI and transient expression, these approaches show that the physical interaction between COP1 and SPA1 was gradually reduced after jasmonate treatment.

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

This manuscript describes an easy and rapid experimental procedure for determining protein-protein interactions based on the measurement of luciferase activity.

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel ...

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation of an adaptive immune response. Experimental work with these cells is rather challenging. Their abundance in organs and tissues is relatively low. As a result, they cannot be isolated in large numbers. They are also difficult to transfect with cDNA constructs. In the murine model, these problems can be partially overcome by in vitro differentiation from bone marrow progenitors in the presence of M-CSF for macrophages or GM-CSF for dendritic cells. In this way, it is possible to obtain large amounts of these cells from very few animals. Moreover, bone marrow progenitors can be transduced with retroviral vectors carrying cDNA constructs during early stages of cultivation prior to their differentiation into bone marrow derived dendritic cells and macrophages. Thus, retroviral transduction followed by differentiation in vitro can be used to express various cDNA constructs in these cells. The ability to express ectopic proteins substantially extends the range of experiments that can be performed on these cells, including live cell imaging of fluorescent proteins, tandem purifications for interactome analyses, structure-function analyses, monitoring of cellular functions with biosensors and many others. In this article, we describe a detailed protocol for retroviral transduction of murine bone marrow derived dendritic cells and macrophages with vectors coding for fluorescently-tagged proteins. On the example of two adaptor proteins, OPAL1 and PSTPIP2, we demonstrate its practical application in flow cytometry and microscopy. We also discuss the advantages and limitations of this approach.

In this article, we provide a detailed protocol for the expression of fluorescent fusion proteins in murine bone marrow derived dendritic cells and macrophages. The method is based on the transduction of bone marrow progenitors with retroviral constructs followed by differentiation into macrophages and dendritic cells in vitro.

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation ...