This work was supported by Fraunhofer USA Center for Molecular Biotechnology, iBio, Inc. and the Defense Advanced Research Projects Agency (grant # HDTRA1-07-C-0054). The authors acknowledge the generous gifts by Drs. Stanton Gelvin of Biological Science Dept., Purdue University (Agrobacterium tumefaciens strains) and Wayne Fitzmaurice of Large Scale Biology Corp. (N. excelsiana seeds), as well as Jennifer Nicholson of US Nicotiana Collection, North Carolina State University (N. excelsior seeds). The authors thank Margaret Shillingford and Christopher Hull for providing plants and excellent technical assistance. The authors also thank Drs. Stephen Streatfield and Natasha Kushnir for editorial assistance.

1. Plant Growing
For subsequent agroinfiltration we evaluated two wild-type Nicotiana species (N. benthamiana and N. excelsior) and a hybrid (N. excelsiana) grown hydroponically on rockwool in indoor facilities.
<ol>
	<li>Soak rockwool slabs in a plant fertilizer solution.</li>
	<li>Sow seeds of wild-type N. benthamiana, N. excelsior and N. excelsiana (hybrid of N. benthamiana &#215; N. excelsior) on the nutrients soaked rockwool surface.</li>
	<li>Grow plants from the seeds under controlled conditions (24 &#176;C and 40-65% relative humidity) and a long-day photoperiod (14 hr light and 10 hr dark, with illumination of 130-150 &#956;E m-2 sec-1) for 4-5 weeks for N. benthamiana and N. excelsiana, and 5-6 weeks for N. excelsior. </li>
</ol>
2. Construction of Vectors for Agroinfiltration
<ol>
	<li>Insert a synthetic reporter gene (green fluorescent protein [GFP]), full-length hemagglutinin (HA) from the A/California/04/2009 strain of influenza virus (HAC1), and re-engineered lichenase enzyme (LicKM)18 separately into the launch vector pBID418 (TMV-based vector) to obtain pBID4-GFP, pBID4-HAC1 and pBID4-LicKM, respectively18,32,41,47.</li>
	<li>Introduce 10-50 ng of pBID4 carrying GFP or HAC1 into electrocompetent cells of A. tumefaciens strain GV3101 and LicKM into electrocompetent cells of A. tumefaciens strains GV3101, C58C1, GLA4404, At06, At10, At77 and A4 with the gene MicroPulser electroporator.</li>
	<li>Use the transformed Agrobacteria for infiltration experiments unless otherwise noted.</li>
</ol>
3. Vacuum Infiltration of Agrobacterium into Nicotiana Plants
<ol>
	<li>Grow A. tumefaciens strains overnight (O/N) in LB medium, YEB medium or AB medium supplemented with 50 mg/L of Kanamycin at 28 &#176;C with shaking at 200-250 rpm.</li>
	<li>Dilute Agrobacteria in Milli-Q water to an optical density at 600 nm (A600) of 0.5 or centrifuge Agrobacterium cells grown in LB or YEB or AB at 4,000 &#215; g for 10 min at 4 &#176;C, re-suspend in induction medium (1x MS salt, 10 mM MES, 200 &#181;M acetosyringone, 2% sucrose [MMA]) to A600 of 0.5, and stir at room temperature for 1-3 hr, unless otherwise noted.</li>
	<li>Infiltrate plants in a vacuum chamber by submerging Nicotiana plant aerial tissues in Agrobacterium suspension and applying a 50-400 mbar vacuum for 30 or 60 sec. The optimal infiltration is routinely applied at 50-100 mbar for 60 sec.</li>
	<li>Once the vacuum is broken, remove plants from the vacuum chamber, rinse in water, and grow for 5-7 days under the same growth conditions used for pre-infiltration growth.</li>
	<li>To test the efficacy of chemicals inducing Agrobacterium vir gene, different concentrations of acetosyringone (0, 100, 200 or 400 &#181;M) were added to the Agrobacteria suspended in infiltration buffer (1x MS, 10 mM MES, 2% glucose). For the effect of monosaccharide on induction of vir gene, different percentages of glucose (0, 1, 2 or 4%) were added to Agrobacteria suspended in the infiltration buffer (1x MS, 10 mM MES, 200 &#181;M acetosyringone). N. benthamiana plants were infiltrated as mentioned above in steps 3.3 and 3.4).</li>
	<li>Agrobacterium laboratory strains GV3101, C58C1 and LBA4404 and wild-type strains A4, At06, At10, and At77 harboring the pBID4-LicKM vector were diluted in Milli-Q water to A600 of 0.5. N. benthamiana plants were infiltrated with each particular strain as mentioned above in steps 3.3 and 3.4.</li>
</ol>
4. Co-agroinfiltration Procedure for the Viral Silencing Suppressor
<ol>
	<li>Mix the Milli-Q water-diluted Agrobacterium GV3101 cultures carrying the GFP gene and the viral silencing suppressor p19 of Tomato bushy stunt virus (TBSV) at 1:1, 2:1, 3:1 and 4:1 ratios. Infiltrate N. benthamiana plants as described above.</li>
	<li>Infiltrate N. benthamiana plants with a mixture of two Milli-Q water-diluted Agrobacterium GV3101 cultures: the first carrying the pBID4-HAC1 plasmid and the second carrying one of the silencing suppressors &#8211; p19 of TBSV or p23 of Citrus tristeza virus, in the pCassp plasmid (pCassp19) and in the pGR binary plasmid under the 35S promoter (pGR-P23), respectively, at the ratio 4:1.</li>
</ol>
5. Western Blot Analysis
<ol>
	<li>Collect random leaf samples from N. benthamiana, N. excelsior or N. excelsiana plants at 4-7 dpi and pulverize in liquid nitrogen to a fine powder.</li>
	<li>Add three volumes of 1x PBS buffer containing 0.5% Triton X-100 to each sample.</li>
	<li>Gently shake the extracted samples for 15 min at 4 &#176;C.</li>
	<li>Spin the extract for 5 min and collect total soluble protein into a clean Eppendorf tube.</li>
	<li>Dilute the extracts to an appropriate dilution (1:50 1:100) in 1x PBS extraction buffer, and add 5&#215; sample buffer (250 mM Tris-HCl [pH 6.8], 10% SDS, 0.5% bromophenol blue, 50% glycerol v/v, and 500 mM DTT) to a final 1&#215; concentration.</li>
	<li>Boil samples for 5 min.</li>
	<li>Separate proteins by 10% SDS-PAGE, transfer onto Immobilon-P transfer membrane, and block with 0.5% I-block.</li>
	<li>Detect GFP using rabbit polyclonal anti-GFP antiserum at 1:5,000 and HAC1 using mouse anti-poly-histidine monoclonal antibody at 1:1,000 in blocking solution for 1 hr.</li>
	<li>After primary antibody labeling, wash the membranes three times for 10 min each with 1&#215; PBST-20 and incubate with a horseradish peroxidase (HRP)-conjugated anti-rabbit antibody at 1:5,000 or a HRP-conjugated anti-mouse antibody at 1:10,000 for 1 hr, for GFP and HAC1 detection, respectively.</li>
	<li>Process Western blots using the SuperSignal West Pico Chemiluminescent substrate.</li>
	<li>Use the GeneTools Software to analyze band intensity of the protein and obtain the band calibrated quantity.</li>
</ol>
Protein production: (Calibrated quantity x dilution of sample)/amount of sample loaded) x 4 = mg/kg. 
Equation: Protein production (P), Calibrated quantity (C), Dilution of the sample (D) and amount of Sample loaded (S).

6. Zymogram Assay
<ol>
	<li>Collect pBID4-LicKM-infiltrated N. benthamiana random tissue samples.</li>
	<li>Extract proteins using the same methods described above for Western blot analysis and then analyze by 10% SDS-PAGE with 0.1% lichenan included in the gels.</li>
	<li>After electrophoresis, wash the gels two times for 10 min each in wash buffer (100 mM Tris-HCl [pH 8.0] and 0.1% Triton X-100) and then incubate in wash buffer at 65 &#176;C for 1 hr.</li>
	<li>After incubation, discard the wash buffer and stain the gels with 0.5% Congo Red for 5 min at room temperature.</li>
	<li>Rinse the gels in Milli-Q water three times for 10 min each, and add 1 M NaCl to visualize lichenase activity. The purified bacterial lichenase protein was used as a positive control for enzyme activity.</li>
</ol>
7. GFP Imaging
<ol>
	<li>Perform visual detection of GFP fluorescence in whole transiently transformed plants using a hand-held long-wavelength UV lamp.</li>
	<li>Photograph transiently transformed plants with a digital camera through a Yellow 8, ES 52 filter (exposure time, 15 sec).</li>
	<li>Obtain images from Western blot analyses using the GeneSnap software on a GeneGnome and quantify the results using the GeneTools software, with a calibration curve based on purified GFP standard.</li>
	<li>Quantify HAC1 protein using a calibration curve based on purified HAC protein standard from the A/Indonesia/05/05 strain of influenza virus.</li>
	<li>Calculate mean values from 3-4 replicates for all experiments.</li>
</ol>

We have nothing to disclose.

In this study, we have developed a simple agroinfiltration protocol for routine transient protein production in selected Nicotiana species using Agrobacterium strains carrying the launch vector. In addition, we have identified the optimum conditions to achieve the highest recombinant protein production level in our transient plant expression system.
Vacuum infiltration of the diluted A. tumefaciens strain GV3101 harboring the launch vector pBID4 into N. benthamiana, N. excelsiana and N. excelsior resulted in higher levels of target protein production within 7 dpi compared to other plant species, such as Pisum sativum infiltrated with GV3101 harboring Alfalfa mosaic virus- or Cucumber mosaic virus-based vectors expressing the GFP reporter gene under the 35S promoter41, or &#160;Lactuca sativa, Solanum lycopersicum and Arabidopsis thaliana infiltrated with the C58C1 strain of A. tumefaciens carrying the beta-glucuronidase reporter gene57. N. benthamiana and N. excelsiana were easy to vacuum infiltrate at 50 mbar for 30-60 sec, with 90-95% infiltration efficiency. The remaining 5-10% of leaf area was not infiltrated because of some floating of leaves on the surface of the Agrobacterium suspension during application of the vacuum. Since the launch vector has ability for cell-to-cell movement18, transient protein accumulation occurs in entire leaves as well as petioles at 7 dpi. At 10 dpi, the estimated GFP production was slightly lower because the pBID4 expression vector is able to move from cell to cell but not to move systemically18; therefore, newly grown leaves do not contain the vector and do not contribute to production of target. In addition, degradation of the recombinant protein over time may contribute to reduced protein level at 10 dpi. Our results showed that infiltration of the A. tumefaciens strain GV3101 mediated high levels of transient protein production in N. benthamiana. Furthermore, target protein can be engineered as N-terminal, C-terminal or internal fusions to lichenase (LicKM), &#946;-1,3-1,4-glucanase, which is a thermostable enzyme from Clostridium thermocellum and confers thermostability to many target protein fusion18. Infiltration of N. benthamiana with A. tumefaciens wild-type strains (at6, at10 and at77) harboring the gene of interest elicited mild or severe symptoms: leaf curling, petiole elongation, and curling. No pathological symptoms were observed in N. benthamiana infiltrated with the laboratory strain GV3101 harboring empty pBID4 vector, while some genes inserted into pBID4 and transformed into laboratory strains GV3101, C58C1 or LBA4404 elicited mild necrotic responses and leaf chlorosis/yellowing symptoms in infiltrated regions of leaves. Necrotic symptoms caused by wild-type Agrobacterium or disarmed strains in solanaceous plants have been reported previously56,57. The necrotic response could result from virulence factors of the Type III secretion system, bacterial proteins transferred to the plant cell by the Type IV secretion system, and/or sensitivity to flagellin58-60. We have found that transient production of heterologous proteins may also elicit pathogenicity and a hypersensitive response in infiltrated plant leaves. Many researchers reported that agroinfiltration of different plant species with plant binary vectors produced up to 5-20 times higher levels of transient protein production compared to stably transformed plants28,57. Our data shows that N. benthamiana infiltrated with GV3101 harboring pBID4-GFP transiently expressed high levels of GFP, which is similar to the GFP yield reported for N. benthamiana infiltrated with Agrobacteria carrying the pICH-GFPSYS viral vector (up to 80% of total soluble protein)44. Agroinfiltration using our launch vector resulted in high production of the thermostable protein LicKM, 50-fold higher than that observed using a standard binary plasmid18.
To test the infectivity of A. tumefaciens and the stability of the launch vector, we infiltrated one flat of N. benthamiana every hour &#160;for up to 8 hr using the same Milli-Q water-diluted culture of GV3101 harboring the pBID4-GFP plasmid. Our data showed that the GV3101 strain is efficiently infective for at least 8 hr and pBID4 (the launch vector) is very stable during the 8 hr-long infiltration.
Glycerol stock of GV3101 strain harboring the launch vector pBID4-HAC1 (cell bank) stored at -80 &#176;C has been shown to be very stable for three years without changes in transient protein production in infiltrated plants. &#160;
N. benthamiana plants grown under optimal conditions and between 35 and 42 days post sowing were optimal for vacuum infiltration-mediated transient gene expression40. Younger plants (3-4 weeks old) cannot be infiltrated entirely because of leaves floating on the cell suspension surface and tissue damage from the mechanical effects of applying a vacuum. In plants older than 45 days, N. benthamiana bolting stage, under the optimal light conditions used, the level of transient expression is low.
Low molecular weight phenolic compounds (acetosyringone) and monosaccharaides (glucose) are known to induce vir genes in A. tumefaciens55,61. Moreover, infiltration of N. benthamiana with the binary vector pCAMBIA(gfp) in the presence of acetosyringone at the concentrations of 50-600 &#181;M were shown to slightly increase transient gene expression40. We studied the effect of different concentrations of acetosyringone and glucose in our system by adding these compounds to GV3101 cultures harboring pBID4-GFP in MMA for 3 hr, and found no difference in GFP expression. Interestingly, GV3101 cultures harboring pBID4-GFP and diluted in Milli-Q water to an A600 of 0.5 and infiltrated without the vir gene induction expressed the same amounts of GFP as those infiltrated with induced cultures. The A. tumefaciens vir gene(s) could potentially be induced by plant phenolic compounds (acetosyringone and sinapinic acid) and plant monosaccharaides (glucose and fructose) present in leaf tissue. Therefore, we speculate that similar levels of GFP expression in the presence or absence of exogenous vir gene inducers may be a result of the effect of cytoplasmic vir gene inducers present during replication of the GFP-expressing launch vector in plant cells.
Wild-type N. benthamiana has been used as a model host for transient protein production49. However, N. benthamiana&#8217;s relatively low biomass yield hinders its application for large-scale production of recombinant proteins. The optimal host should combine a high level of transient expression, easy growth in a greenhouse, and be susceptible to Agrobacterium infiltration2. To select an alternative host, we infiltrated two different wild-type species of Nicotiana (N. benthamiana and N. excelsior) and a hybrid N. excelsiana (N. benthamiana &#215; N. excelsior) with the A. tumefaciens strain GV3101 harboring the pBID4-GFP plasmid. Among these three species, the level of GFP expression was slightly higher in N. benthamiana. N. excelsior plants showed difficulty in vacuum agroinfiltration due to their leathery leaves, and N. excelsiana produced approximately two-fold more biomass under the same growth conditions. The transient production of GFP at 7 dpi is relatively similar in N. benthamiana and N. excelsiana. Therefore, N. excelsiana may be a more suitable host for recombinant protein production.
Agrobacterium-mediated transient protein production is limited by PTGS26, which can be overcome by co-expression of gene silencing suppressors of plant virus origin62. Transient protein production has been previously shown to be enhanced 50-fold in the presence of the p19 protein of TBSV, which inhibits PTGS in infiltrated tissues34. In our study, we assessed the effect of two viral silencing suppressors (p19 and p23) separately co-infiltrated with the launch vector pBID4-HAC1. The co-infiltration of these silencing suppressors seemed to have little influence on transient expression of HAC1, with only a slight increase in HAC1 protein accumulation (15-25%) in the presence of co-infiltrated p23 or p19. To positively affect protein production, silencing suppressors may need to be specifically selected to be effective for the targeted plant species and viral vector63. TMV helicase has a suppressor of RNA silencing activity64,65. Our data confirm this observation as co-infiltration of p23 or p19 with pBID4-HAC1 resulted in no increase in GFP or a slight increase in the transient HAC1 protein production.
In conclusion, we have modified and optimized plant and Agrobacterium growth conditions and improved the efficiency of vacuum infiltration. This technology enabled us to grow and infiltrate hundreds of kilograms of plant material in a few hours. We successfully automated the plant transient expression technique for high-throughput vaccine production at industrial scales under current Good Manufacturing Practices (cGMP) conditions. For more information about automation and utilization of the plant transient protein production system for the production of recombinant proteins, including subunit vaccine candidates, under cGMP conditions, readers are referred to the website <a href="http://fhcmb.org/">www.fhcmb.org/</a>.

Plants are now recognized as a safe, reliable, scalable and inexpensive platform for producing heterologous recombinant biopharmaceuticals and industrial proteins1-3 and have important advantages over microbial and animal cell expression systems4. Plants are able to express correctly folded proteins with post-translational modifications, including assembled multimeric antibodies5-7. Several plant-derived recombinant pharmaceutical proteins are undergoing clinical evaluation8. These include patient-specific recombinant idiotype vaccines (scFv) for the treatment of non-Hodgkin&#8217;s lymphoma9, hemagglutinin-based pandemic and seasonal influenza vaccine candidates10,11 (Cummings et al., submitted to Vaccine), anti-Streptococcus surface antigen I/II antibody for the treatment of dental caries12, and human insulin for the treatment of diabetes13. Furthermore, human recombinant glucocerebrosidase for enzyme replacement therapy in patients with Gaucher disease has been approved in Israel and the US and is provided under the Expanded Access Program outside of the US14,15.
Heterologous proteins can be produced in stably transformed (transgenic or transplastomic) or transiently transformed plants. Transient protein production offers several advantages over production in transgenic plants, including short timeframe to achieve expression and accumulation16, and can be achieved by introducing bacterial binary vectors or recombinant plant viral vectors into plant tissues4. The most advanced transient expression system is based on the use of &#8216;launch vectors&#8217; that combine components of plant viruses and binary plasmids, and are delivered by agroinfiltration17,18. Agroinfiltration of a launch vector based on Tobacco mosaic virus (TMV) has been successfully applied at lab scale to produce vaccine antigens against pathogens such as human papilloma virus19, Yersinia pestis20, influenza viruses A21,22, Bacillus anthracis23, and smallpox virus24 in N. benthamiana leaves. Agrobacterium-mediated transient expression is also a promising method for the simultaneous production of multiple proteins2,25-27. For example, plant transient expression systems have been used to produce tumor-specific recombinant antibodies28,29, a glycosylated recombinant antibody against the epidermal growth factor receptor30, and a monoclonal antibody specific for anthrax protective antigen31,32. Co-infiltration of Nicotiana benthamiana plants with a target gene and a suppressor of gene silencing results in enhanced target protein expression33,34.
Agroinfiltration is a common method for uniformly introducing bacteria harboring a gene of interest into plant tissues35-37. Vacuum infiltration of Agrobacterium for transient gene expression in intact plant leaves is a rapid, scalable, and useful method for production of foreign proteins without the need to generate transgenic plants38-41. During vacuum agroinfiltration, plants are flipped upside down and aerial parts submerged in Agrobacterium suspension. Then vacuum is applied causing gases to evacuate from leaf intercellular spaces through stomata. Rapid re-pressurization following release of the vacuum results in the infusion of the Agrobacterium suspension into the leaf. Following vacuum infiltration of Agrobacteria, plants are further cultivated and target expression is monitored. The highest levels of target expression are typically observed 2-3 days post infiltration (dpi) with a binary vector and 4-7 dpi with a launch vector, after which the expression level typically decreases17,18,42-45. Agrobacterium tumefaciens is the most widely used vehicle for delivering a gene of interest into a plant for protein production. Agroinfiltration works exceptionally well in N. benthamiana but relatively poorly in most other plants, including Arabidopsis thaliana46.
In this study, we developed a simple, efficient, and economical method for transient protein production in 5&#8211;6 week-old N. benthamiana using A. tumefaciens infiltration. The major drawback of industrial scaling of the agroinfiltration technique is centrifugation of harvested bacteria and resuspension of the bacterial pellet in medium containing 4'-hydroxy-3',5'-dimethoxyacetophenone (acetosyringone), monosaccharides, and 2-(N-morpholino)-ethanesulfonic acid (MES) buffer for induction of the vir genes. We have been able to overcome these problems by optimizing the Agrobacterium growth in AB medium (minimal medium) followed by directly diluting in Milli-Q water and by controlling the infiltration duration and conditions. We have also compared target protein production in the wild-type tobacco host species N. benthamiana and N. excelsior, as well as in hybrid N. excelsiana.

<table border="0" cellpadding="0" cellspacing="0" width="792">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Nicotiana benthamiana</td>
			<td style="width:221px;">Tobacco Germplasm Collection, Crop Science Dept., North Carolina State University</td>
			<td style="width:119px;">PI 555478 TW16</td>
			<td style="width:236px;">Infiltration</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Nicotiana excelsior</td>
			<td style="width:221px;">Tobacco Germplasm Collection, Crop Science Dept., North Carolina State University</td>
			<td style="width:119px;">PI 555685 TW47</td>
			<td style="width:236px;">Infiltration</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Nicotiana excelsiana</td>
			<td style="width:221px;">Dr. Wayne Fitzmaurice, Large Scale Biology Corporation, Vacaville, CA</td>
			<td style="width:119px;">LSBC EBA 042304.02</td>
			<td style="width:236px;">Infiltration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vacuum skid</td>
			<td style="width:221px;">Abbas, Ashland, MA</td>
			<td style="width:119px;">Custom made</td>
			<td style="width:236px;">Plant infiltration</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Rockwool</td>
			<td style="width:221px;">Grodan Inc., Ontario, Canada</td>
			<td style="width:119px;">AO 50/40</td>
			<td style="width:236px;">Hydroponic media for growing plant</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">2-(N-Morpholino) ethanesulfonic acid</td>
			<td style="width:221px;">Acros Organics, Thermo Fisher Scientific&#160; 
			NJ</td>
			<td style="width:119px;">172595000</td>
			<td style="width:236px;">Buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Murashige &#38; Skoog salt (MS salt)</td>
			<td style="width:221px;">Phyto Technology Lab</td>
			<td style="width:119px;">M524</td>
			<td style="width:236px;">Tissue culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acetosyringone</td>
			<td style="width:221px;">Sigma-Aldrich, St. Louis, MO</td>
			<td style="width:119px;">D134406-5G</td>
			<td style="width:236px;">Agrobacterium induction</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Immobilon-P transfer membrane</td>
			<td style="width:221px;">Millipore, Billerica, MA</td>
			<td style="width:119px;">IPVH00010</td>
			<td style="width:236px;">Western blotting</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">I-block</td>
			<td style="width:221px;">Applied Biosystems, Foster City, CA</td>
			<td style="width:119px;">T2015</td>
			<td style="width:236px;">Western blotting</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Rabbit polyclonal anti-GFP antiserum&#160;</td>
			<td style="width:221px;">Washington Biotechnology, Baltimore, MD&#160;&#160;&#160;&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:236px;">Western blotting</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Mouse anti&#8211;poly-histidine monoclonal antibody</td>
			<td style="width:221px;">Qiagen GmbH, Hilden</td>
			<td style="width:119px;">34670</td>
			<td style="width:236px;">Western blotting</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Horseradish peroxidase-conjugated anti-rabbit antibody&#160;</td>
			<td style="width:221px;">Jackson ImmunoResearch, West Grove, PA</td>
			<td style="width:119px;">111-035-003</td>
			<td style="width:236px;">Western blotting</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Horseradish peroxidase-conjugated anti-mouse antibody</td>
			<td style="width:221px;">Jackson ImmunoResearch, West Grove, PA</td>
			<td style="width:119px;">115-035-003</td>
			<td style="width:236px;">Western blotting</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">SuperSignal&#174; West Pico chemiluminescent substrate</td>
			<td style="width:221px;">Thermo Scientific Pierce, Rockford, IL</td>
			<td style="width:119px;">34078</td>
			<td style="width:236px;">Western blotting</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Lichenan (1-3: 1-4-beta-D-glucan)</td>
			<td style="width:221px;">Megazyme, Bray, Co. Wicklow, Ireland&#160;</td>
			<td style="width:119px;">P-LICHN</td>
			<td style="width:236px;">Lichenase Activity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Congo Red&#160;</td>
			<td style="width:221px;">Sigma-Aldrich, St. Louis, MO</td>
			<td style="width:119px;">C6277&#160;</td>
			<td>Gel staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Digital camera&#160;</td>
			<td style="width:221px;">Olympus, Center Valley, PA</td>
			<td style="width:119px;">C-8080</td>
			<td>Chemiluminescence imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GeneGnome</td>
			<td style="width:221px;">Syngene, Frederick, MD</td>
			<td style="width:119px;"></td>
			<td>Chemiluminescence imaging&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GFP standard&#160;</td>
			<td style="width:221px;">Made in house&#160;</td>
			<td style="width:119px;"></td>
			<td>Chemiluminescence imaging&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Plant fertilizer solution</td>
			<td style="width:221px;">Griffin Greenhouse &#38; Nursery Supplies, Newark, DE</td>
			<td style="width:119px;">67-23-20</td>
			<td>Plant growing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lichenase Standard</td>
			<td style="width:221px;">Purified in house&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:236px;">Western blotting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MicroPulser Electroporator</td>
			<td style="width:221px;">BioRad, Hercules, CA&#160;</td>
			<td>165-2100</td>
			<td>Agrobacterium transformation&#160;</td>
		</tr>
	</tbody>
</table>

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).

Watch this Scientific Journal Video about Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana at JoVE.com

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.

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 ...

optimization-utilization-agrobacterium-mediated-transient-protein

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JoVE Journal

Biology

41.0K Views.  Fraunhofer USA Center for Molecular Biotechnology. 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.

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

Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells

Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, Bcr-PDGFR&#945;, etc.). We are able to model and study a human-like disease in our mouse model, by transplanting bone marrow cells previously infected with a retrovirus expressing the oncogene of interest. Replication-defective retrovirus encoding a human oncogene and a marker (GFP, RFP, antibiotic resistance gene, etc.) is produced by a transient transfection protocol using 293T cells, a human renal epithelial cell line transformed by the adenovirus E1A gene product. 293 cells have the unusual property of being highly transfectable by calcium phosphate (CaPO4), with up to 50-80% transfection efficiency readily attainable. Here, we co-transfect 293 cells with a retroviral vector expressing the oncogene of interest and a plasmid that expresses the gag-pol-env packaging functions, such as the single-genome packaging constructs kat or pCL, in this case the EcoPak plasmid. The initial transfection is further improved by use of chloroquine. Stocks of ecotropic virus, collected as culture supernatant 48 hrs. post-transfection, can be stored at -80&deg;C and used for infection of cell-lines in view of transformation and in vitro studies, or primary cells such as mouse bone marrow cells, that can then be used for transplant in our mouse model.

This technique demonstrates an efficient way to prepare replication-defective retroviral stocks encoding a human oncogene, and subsequently used for induction of myeloproliferative disease in the mouse model.

Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, ...

A Method For Production of Recombinant mCD1d Protein in Insect Cells.

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated heavy chains that are noncovalently associated with beta2-microglobulin. They bind lipids rather than peptides. Although their structure confirms the similarity of CD1 proteins to MHC class I and class II antigen presenting molecules, the mCD1d groove is relatively narrow, deep, and highly hydrophobic and it has two binding pockets instead of the several shallow pockets described for the classical MHC-encoded antigen-presenting molecules. Based upon their amino acid sequences, such a hydrobphobic groove provides an ideal environment for the binding of lipid antigens. The Natural Killer T (NKT) cells use their TCR to recognize glycolipids bound to or presented by CD1d. T cells reactive to lipids presented by CD1 have been involved in the protection against autoimmune and infectious diseases and in tumor rejection. Thus, the ability to identify, purify , and track the response of CD1-reactive NKT cell is of great importance . The generation of tetramers of alpha Galactosyl ceramide (a-Galcer) with CD1d has significant insight into the biology of NKT cells. Tetramers constructed from other CD1 molecules have also been generated and these new reagents have greatly expanded the knowledge of the functions of lipid-reactive T cells, with potential use in monitoring the response to lipid-based vaccines and in the diagnosis of autoimmune diseases and other treatments.

A Method to prepare Insect cells and infect them with baculovirus for the the purpose of production of recombinant mCD1d proteinand generating mCD1d tetramers.

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions.
Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation.
In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.

We present the detailed protocol for Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in cotton. The tobacco rattle virus (TRV)-derived VIGS vectors were deployed to induce RNA silencing of cotton GrCLA1, Cloroplastos alterados 1 gene. The albino phenotype caused by silencing GrCLA1 was observed at the seedling stage within 2 weeks after inoculation.

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

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 ...

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 ...

Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes

Demonstrated here is a detailed protocol for Agrobacterium-mediated genetic transformation of maize inbred lines using morphogenic genes Baby boom (Bbm) and Wuschel2 (Wus2). Bbm is regulated by the maize phospholipid transferase gene (Pltp) promoter, and Wus2 is under the control of a maize auxin-inducible (Axig1) promoter. An Agrobacterium strain carrying these morphogenic genes on transfer DNA (T-DNA) and extra copies of Agrobacterium virulence (vir) genes are used to infect maize immature embryo explants. Somatic embryos form on the scutella of infected embryos and can be selected by herbicide resistance and germinated into plants. A heat-activated cre/loxP recombination system built into the DNA construct allows for removal of morphogenic genes from the maize genome during an early stage of the transformation process. Transformation frequencies of approximately 14%, 4%, and 4% (numbers of independent transgenic events per 100 infected embryos) can be achieved for W22, B73, and Mo17, respectively, using this protocol.

Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes

Plant morphogenic genes can be used to improve genetic transformation of recalcitrant genotypes. Described here is an Agrobacterium-mediated genetic transformation (QuickCorn) protocol for three important public maize inbred lines.

Demonstrated here is a detailed protocol for Agrobacterium-mediated genetic transformation of maize inbred lines using morphogenic genes Baby boom (Bbm) ...

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice

Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive organs/gametes. Recent advances in CRISPR-Cas9 genome editing technology allow for high editing efficacy and timesaving knockout mutations of endogenous candidate genes at specific sites. Additionally, Agrobacterium-mediated genetic transformation of rice is also a key method for gene modification, which has been widely adopted by many public and private laboratories. In this study, we applied CRISPR-Cas9 genome editing tools and successfully generated three male sterile mutant lines by targeted genome editing of OsABCG15 in a japonica cultivar. We used a modified Agrobacterium-mediated rice transformation method that could provide excellent means of genetic emasculation for hybrid seed production in rice. Transgenic plants can be obtained within 2&#8211;3 months and homozygous transformants were screened by genotyping using PCR amplification and Sanger sequencing. Basic phenotypic characterization of the male sterile homozygous line was performed by microscopic observation of the rice male reproductive organs, pollen viability analysis by iodine potassium iodide (I2-KI) staining semi-thin cross-sectioning of developing anthers.

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice

This work describes the use of CRISPR-Cas9 genome editing technology to knockout endogenous gene OsABCG15 followed by a modified Agrobacterium-mediated transformation protocol to produce a stable male-sterile line in rice.

Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive ...

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.

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.

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