We thank R. Sun and other students of Chen's Laboratory for their contribution to plant material generation. We also thank Dr. D. Green for his support of undergraduate research in the College of Technology and Innovation (CTI). This research was supported in part by NIH grants U01 AI075549 and 1R21AI101329 to Q. Chen, and a SSE grant from CTI of Arizona State University to Q. Chen. K. Leuzinger, M. Dent, J. Hurtado, and J. Stahnke are undergraduate students supported by the SSE grant.

1. Plant Growth
<ol>
	<li>Place 60 peat pellets into a propagation tray. Add 4 L of tap water and let the peat pellets absorb the water for 2 hr.</li>
	<li>Add 2 N. benthamiana seeds into each peat pellet using a seeder, cover the tray with a transparent plastic dome, and allow them to germinate in a 25 &deg;C, 84% humidity environment with a 16/8 hr day/night cycle (Figure 1A).</li>
	<li>Remove the dome two weeks after seeding, drain water from the tray, and add 2 L of Jack's fertilizer at a concentration of 1.48 g/L (Figure 1B). Continue to grow plants in a 25 &deg;C, 50% humidity environment with a 16/8 hr day/night cycle. Supply 2 L of Jack's fertilizer every 2 days per tray.</li>
	<li>At week 4, transfer the plants with peat pellets to a new tray that hosts six plants to provide adequate space for further growth (Figure 1C) until they are ready to be infiltrated at 6 weeks of age (Figure 1D).</li>
</ol>
2. A. tumefaciens Culture Preparation
2.1 Preparation for Syringe Infiltration
<ol>
	<li>Streak A. tumefaciens GV3101 strains containing the geminiviral vectors p19, pREP110, pBYGFP, and pBYDsRed on LB Agar plates containing kanamycin (100 &mu;g/ml). Streak one strain per plate and grow at 30 &deg;C for 48 hr.</li>
	<li>Similarly, streak and grow GV3101 strains harboring the MagnICON vectors of the 5' module (pICH15879), the 3' module containing the target gene of GFP or DsRed (pICH-GFP or pICH-DsRed), and Integrase (pCH14011) on LB Agar plates containing carbenicillin (100 &mu;g/ml).</li>
	<li>From a single colony on the LB Agar plate, inoculate GV3101 strains harboring geminiviral vectors into 3 ml of YENB media (0.75% Bacto yeast extract, 0.8% Nutrient Broth, adjust pH to 7.5 with NaOH) + kanamycin (100 &mu;g/ml) in a 15 ml round-bottom culture tube, grow the liquid culture at 30 &deg;C in a shaker overnight with a 300 rpm rotation rate.</li>
	<li>Similarly, inoculate and grow GV3101 strains harboring MagnICON vectors in YENB media + carbenicillin (100 &mu;g/ml) in a shaker overnight at 30 &deg;C.</li>
	<li>Measure and record OD600 values for each liquid culture with a spectrophotometer and use the formula below to calculate the necessary volume (Vsub) to be subcultured for a new culture with a starting OD600 of 0.025 in 10 ml of YENB media with appropriate antibiotics. 
	Vsub (ml) = (10 ml) x (0.025) / OD600</li>
	<li>Transfer Vsub (ml) of the overnight culture to (10 - Vsub) ml of YENB media with appropriate antibiotics for each strain. Grow the new culture overnight at 30 &deg;C in a shaker with a 250 rpm rotation rate until the OD600 value is in the range of 1.7-2.0.</li>
	<li>Measure and record OD600 values for each liquid culture and use the formula below to calculate the necessary volume (Vinf) to be diluted in 50 ml of infiltration buffer to give a final OD600 of 0.12 for each strain. 
	Vinf (ml) = (50 ml) x (0.12) / OD600</li>
	<li>Transfer Vinf (ml) of each culture to a microcentrifuge tube and spin down the cells by centrifugation at 12,000 x g for 2 min, remove supernatant and then resuspend the cells in 10 ml infiltration buffer (10 mM MES, pH 5.5; 10 mM MgSO4) by vortexing briefly.</li>
	<li>Mix resuspended cells of pBYGFP + pREP110 + p19, spin the cells down at 12,000 x g for 2 min, and resuspend in total of 50 ml of infiltration buffer. This Agrobacteria combination is for geminiviral expression of GFP.</li>
	<li>Similarly, mix the following combinations of Agrobacteria cells, spin down and resuspend them in 50 ml of infiltration buffer. They include (1) pBYDsRed + pREP110 + p19 for geminiviral expression of DsRed, (2) pBYGFP + pBYDsRed + pREP110 + p19 for geminiviral co-expression of GFP and DsRed, (3) pREp110 + p19 as negative control of geminiviral expression, (4) pICH-GFP + pICH15879 + pCH14011 for MagnICON expression of GFP, (5) pICH-DsRed + pICH15879 + pCH14011 for MagnICON expression of DsRed, and (6) pICH15879 + pCH14011 as negative control for MagnICON expression.</li>
</ol>
2.2 Preparation for Vacuum Infiltration
<ol>
	<li>Inoculate and culture each GV3101 strain containing geminiviral vectors or MagnICON vectors into 15 ml of YENB media with appropriate antibiotics in a 50 ml autoclaved flask and grow overnight as described for syringe infiltration above.</li>
	<li>Measure and record OD600 values for each liquid culture with a spectrophotometer and use the formula below to calculate the necessary volume (Vsub) to be subcultured for a new culture with a starting OD600 of 0.025 in 250 ml of YENB media with appropriate antibiotics. 
	Vsub (ml) = (250 ml) x (0.025) / OD600</li>
	<li>Transfer Vsub (ml) of the overnight culture to (250 - Vsub) ml of YENB media in a 1 L autoclaved flask with appropriate antibiotics for each strain and grow the new culture overnight as described for syringe infiltration above.</li>
	<li>Measure and record OD600 values for each liquid culture and use the formula below to calculate the necessary volume (Vinf) to be diluted in 3 L of infiltration buffer to give a final OD600 of 0.12 for each strain. 
	Vinf (ml) = (3000 ml) x (0.12) / OD600</li>
	<li>Pellet and resuspend Vinf (ml) of each culture in infiltration buffer as described for syringe infiltration and mix the resuspended cultures according to the combinations described for syringe infiltration. Repellet the mixed Agrobacteria cells and resuspend them in 3 L of infiltration buffer.</li>
</ol>
3. Infiltration
3.1 Syringe Infiltration
<ol>
	<li>Choose four 6-week old N. benthamiana plants with 5 leaves each. The first three leaves counting from the top of each plant will be infiltrated entirely with one of the combinations of A. tumefaciens strains in infiltration buffer. The fourth leaf will be spot-infiltrated with all four strain combinations for geminiviral expression or all three combinations for MagnICON expression. The last leaf on each plant will be infiltrated with combinations of negative controls.</li>
	<li>Create a small nick with a needle in the epidermis on the back side of the leaf (Figure 2A). Attention: make sure not to scratch so hard as to pierce the leaf through both sides, as the Agrobacteria cells in the infiltration mixture will pass through the puncture to the other side of the leaf.</li>
	<li>Take a firm hold of the front side of the leaf and while applying gentle counter pressure to the nick with the thumb of one hand, inject the Agrobacterium mixtures in infiltration buffer into the nick with a syringe without a needle (Figure 2B). Note: As the Agrobacterium mixture enters the intercellular space of the leaf, the infiltrated area will turn visibly darker green (Figure 2C).</li>
	<li>Continue to inject the Agrobacterium mixtures into the nick until the darker green circle stops to expand. Create another nick and repeat steps 3.1.2-3.1.4 until the entire leaf is infiltrated and the whole leaf turns darker green for the first three leaves and the last leaf.</li>
	<li>For the fourth leaf of each plant, all four (geminiviral vector) or three (MagnICON vector) combinations will be infiltrated into it. Make one nick for each combination of Agrobacterium strains, and infiltrate each nick with one combination.</li>
	<li>After infiltration, move plants back to the growth room and monitor protein expression between 2-15 days post infiltration (dpi).</li>
</ol>
3.2 Vacuum Infiltration
<ol>
	<li>Place a tub into a vacuum desiccator and transfer 3 L infiltration buffer containing the Agrobacterium strains to the tub. Connect the desiccator to a Vacuubrand diaphragm vacuum pump (Figure 3A).</li>
	<li>Place a plant upside down on the desiccator plate (Figure 3B) and lower the plate with the plant until the entire leaf and stem system is submerged into the infiltration buffer with the plate resting atop of the tub. Place the desiccator O ring along the rim and put the desiccator lid on the chamber (Figure 3C).</li>
	<li>Turn on the vacuum pump and start timing when the vacuum reaches 100 mbar. Slowly open the release valve on the desiccator after 1 min at 100 mbar to allow entrance of Agrobacteria into the interstitial spaces of submerged plant tissue. Repeat this step one additional time to ensure good infiltration.</li>
	<li>After infiltration, take the plant out of the desiccator and put it back to its upright position. Move plants back to the growth room and monitor protein expression between 2-15 dpi.</li>
</ol>
4. Fluorescent Protein Detection and Photography
<ol>
	<li>Starting from 2 dpi, move plants into dark room and shine UV light on the back side of infiltrated leaves with a hand-held UV lamp.</li>
	<li>Observe the green fluorescence of GFP or the red fluorescence of DsRed. GFP gives off a stronger signal with long wave UV light, while DsRed is stronger under short wave UV light.</li>
	<li>Take photographs of fluorescent leaves with a regular digital camera with no flash.</li>
	<li>Move plants back to the growth room after observation or photography.</li>
</ol>

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The authors have no competing financial interests.

The increasing demands for protein-based pharmaceuticals worldwide require new production platforms that are robust, scalable, low-cost and safe. Plants have shown to be one of the most promising alternative production systems for pharmaceutical protein production. In recent years, the development of deconstructed virus-based vectors has enabled transient expression of proteins in plants, which greatly enhances the speed and yield of plant expression systems 2,10. To further optimize the utility of the transient expression system, we demonstrate a simple yet efficient and scalable approach to introduce target-gene containing Agrobacterium into plant tissue. Our results indicate that agroinfiltration with either the syringe or vacuum method has resulted in the efficient introduction of Agrobacterium into the plant leaves and robust production of two fluorescent proteins, GFP and DsRed.
To ensure the efficient agroinfiltration and protein production, the following critical parameters must be carefully controlled. Deviations from these parameters may result in low agroinfiltration efficiency and in turn, low target protein expression.
1. Plant developmental stage and health. The most likely variable in this method between laboratories is the plant material for infiltration. While plants may appear phenotypically similar, their developmental stage and physiological state affect their competency in expressing recombinant proteins significantly. Specific parameters which have impacts on plant growth and protein expression levels include temperature, humidity, light intensity, supply of fertilizer, plant inoculation age, and time required for the maximum accumulation of target protein after leaf infiltration. Plants must consistently receive equal amounts of water and fertilizer daily. Minor changes in the plant growth conditions may drastically change the final size of plants and their ability to express recombinant proteins. Our previous studies indicate that plants grown under natural light yielded more leaf biomass, but protein yield is much less than that grown under artificial light 4. Therefore, using artificial light is the method of choice for plant growth. Our results also show that a 16 hr light/8 hr dark cycle at 25 &plusmn; 0.5 &deg;C is the optimal condition to grow N. benthamiana plants under such artificial lighting 4. We demonstrated that under these conditions, 6-week plants are the optimal age for GFP and DsRed expression as they produce high-levels of fluorescent proteins while the biomass yield is adequate. Plants older than 6-weeks produce more biomass but are too tall to fit into the infiltration chamber 4. In addition, flowers start to develop after 6 weeks of growth, negatively affecting recombinant protein expression 4. Consequently, 6-week plants contain the optimal leaf material that balances the combined need of biomass yield, protein accumulation, and the ease of agroinfiltration.
2. Growth and infiltration concentration of Agrobacterium. Another key point in this methodology is the control of growth and infiltration concentration of A. tumefaciens, as measured by the OD600. In each culture and subculture step, strains of A. tumefaciens must be grown to, but not over the designated OD600. We have examined multiple concentrations of A. tumefaciens for the final infiltration of N. benthamiana leaves 4. Low Agrobacterium concentration will result in the insufficient delivery of target genes into plants, leading to low protein expression. On the other hand, if the infiltration concentration of Agrobacterium is too high, it will trigger a hypersensitive response in the infiltrated tissue and lead to necrosis 20. Our results demonstrated that OD600 = 0.12 Agrobacterium strain balances the need for maximum delivery of gene construct without causing tissue necrosis and cell death. The desired OD600 density of Agrobacterium can be obtained by using consistent culture media, temperature and culture time.
3. For vacuum infiltration, it is important to follow the designated vacuum pressure and infiltration duration. Our results indicate that one 3 L tub of Agrobacterium infiltration mixture can be used to infiltrate approximately 30 plants. If more than 30 plants need to be infiltrated, a new batch of Agrobacterium infiltration mixture needs to be supplied.
4. The specific parameters presented in this paper are optimized for expression of proteins using N. benthamiana plants grown under specific conditions described above with deconstructed virus-based vectors. As discussed earlier, the most difficult parameter to control in this methodology is the plant material. We have expressed a variety of vaccines and therapeutic proteins using plants and the infiltration procedure we described here and obtained excellent results in all cases 4,8,14,18,21-23. These results demonstrated that the conditions we have developed are optimal for protein expression. However, if different host plant species, expression vectors, or different N. Benthamiana growing conditions were used for infiltration, each parameter in this methodology would need to be re-optimized by experiments.
Overall, we have demonstrated that agroinfiltration with syringe and vacuum is a simple yet efficient methodology to introduce target gene carrying Agrobacterium into plants for transient expression of recombinant proteins. Both methods have their unique advantages depending on the goal of research and production. Syringe infiltration is simple, requires only small volumes of Agrobacterium culture and does not need expensive pumps and vacuum chambers. As demonstrated in this report, it has the flexibility to either infiltrate the whole leaf with one target gene or use spot infiltration to introduce genes of multiple targets on one leaf. The entire leaf infiltration method can be used for small laboratory scale expression of recombinant protein for their biochemical characterization, purification, and preclinical functional studies 1. In contrast, spot infiltration can be used to express multiple protein targets on one leaf to compare their yield, expression kinetics and toxicity to plants. It can also be used to compare the expression of one reporter protein such as GFP or DsRed driven by different expression vectors on the same leaf. As a result, different vector's ability in driving protein accumulation and their expression kinetics can be characterized. The simplicity of syringe infiltration also enables it a feasible tool to teach and train high school and undergraduate students in the subject of biotechnology and genetic engineering.
In comparison with syringe infiltration, vacuum infiltration requires the investment of vacuum pumps and chambers and larger volumes of Agrobacterium cultures. Therefore, it is not the method of choice when just a few plants need to be infiltrated. However, it provides the scalability that cannot be matched by syringe infiltration. It is more robust and can infiltrate large numbers of plants in a short period of time. With the scale presented in this report, we are able to produce gram level of purified pharmaceutical proteins sufficient for a Phase I human clinical trial 4. Furthermore, this process can be further scaled-up for commercial manufacture of pharmaceutical proteins from plants. For example, processes are being designed to vacuum infiltrate three metric tons of N. benthamiana plants per hour in a scale-up operation 24. Another advantage of vacuum infiltration is that it can be used to agroinfiltrate plant species that are not amenable for syringe infiltration.
In summary, the combination of syringe and vacuum infiltration provides researchers, biotechnologists and educators a simple, efficient, robust and scalable methodology for transient expression of recombinant proteins in plants. It will greatly facilitate the development and production of pharmaceutical proteins and promote science education.

Since the 1970s, plants have been explored as alternatives to mammalian, insect, and bacterial cell cultures for the commercial production of recombinant proteins and protein therapeutics 1. Plant-based systems for the expression of biopharmaceuticals have shown promise in recent years as several novel treatments for diseases, like Gaucher's Disease 2, and avian H5N1 influenza 3, have shown success in clinical trials. The development of competent mechanisms for recombinant protein expression in plants in the decades since those initial experiments has created the potential for plant-based systems to alter the current paradigm of protein production for three primary reasons. Firstly, there is a notable decrease in cost as mammalian, insect, and bacterial bioreactors require considerable startup costs, expensive growth media, and complicated processes for downstream purification 4. The creation of stable transgenic plant lines also allows them to outpace the scalability of other expression systems as protein expressing plants could be grown and harvested on an agricultural scale 5. Secondly, plant-based expression systems significantly reduce the risk of transmitting a human or animal pathogen from the protein-expressing host to humans, demonstrating superiority in public safety 6. Lastly, plants utilize a eukaryotic endomembrane system that is similar to mammalian cells, allowing for proper post-translational modification of proteins including glycosylation and the assembly of multiple-subunit proteins 7. This ability puts plant-based systems ahead of those based on prokaryotic systems, such as bacteria, since a wider number of pharmaceutical recombinant proteins, including monoclonal antibodies (mAbs), have a more complicated structure and require extensive posttranslational modifications or assembly 8.
There are two major approaches to expressing recombinant proteins in plants. The first is the development of a stably transgenic line, where DNA coding for the target protein is cloned into an expression cassette and introduced to either the nuclear or chloroplast genomes. In doing so, the foreign DNA becomes heritable through succeeding generations and allows for tremendously improved scalability, far beyond that of other expression systems 1. Introduction of exogenous DNA to the nuclear genome is usually achieved by Agrobacterium tumefaciens infection of plant tissue or, less often, by microprojectile bombardment of the tissue 9. Plant hormones are then used to induce differentiation and growth of transgenic plant tissue such as roots and leaves. Transformation of the chloroplast genome cannot be achieved with A. tumefaciens, but relies entirely on gold or tungsten particles coated with DNA fired ballistically into plant cells. The second method of expressing recombinant protein in plants is through transient expression 10. In this scenario, virus-derived vectors harboring the gene of interest are delivered via A. tumefaciens to fully developed plants through a process called agroinfiltration. Instead of integrating into the plant genome, the delivered gene construct will then begin to direct the transient production of the desired protein, which can be harvested and isolated after a short incubation period. Transient gene expression offers the advantage of greater overall protein accumulation as well as an improved time of protein production, as plants will be ready to harvest approximately 1-2 weeks after agroinfiltration 11. This is significantly faster than the processes of generation, selection, and confirmation of stable transgenic plant lines, which can take several months to a year. This however, is also the limitation of the transient expression system, as it will not yield genetically stable plant lines that can be used to generate a seed bank for large scale commercial production. Despite this, approaches have been developed to improve large scale transient expression. Here we demonstrate one method of generation of transient protein-expressing Nicotiana benthamiana plants using deconstructed viral vectors delivered by A. tumefaciens.
Two major methods are being developed for the delivery of A. tumefaciens into plant tissue: bench scale infiltration via syringe and large scale infiltration via vacuum chamber. Both protocols are described here using N. benthamiana, which is closely related to the common tobacco plant, as the host plant for transient expression of two fluorescent proteins: the green fluorescent protein (GFP) from jellyfish Aequorea victoria and the red fluorescent protein from Discosoma coral (DsRed) 12,13. N. benthamiana is the most common host plant for recombinant protein because it is amenable to genetic transformation, can yield high amounts of biomass rapidly, and is a prolific seed producer for scale-up production 14. Another advantage of using N. benthamiana as hosts for protein expression is the availability of a variety of expression vectors 2,5. In this study, two deconstructed viral vectors, one based on a tobacco mosaic virus (TMV) RNA replicon system (MagnICON vectors) and the other derived from the bean yellow dwarf virus (BeYDV) DNA replicon system (geminiviral vectors) 4,11,15-18 , are used to carry the GFP and DsRed gene and deliver them into N. benthamiana cells via A. tumefaciens. Three DNA constructs will be used for GFP or DsRed expression with MagnICON vectors. They include the 5' module (pICH15879) containing the promoter and other genetic elements for driving the expression of the target gene, the 3' module containing the gene of interest (pICH-GFP or pICH-DsRed), and the integrase module (pICH14011) coding for an enzyme that integrates the 5' and 3' modules together upon expression 8,15. Three DNA constructs are also needed for expression with geminiviral vectors. In addition to vectors containing the replicon of the target gene ( pBYGFP or pBYDsRed), a vector coding for the replication protein (pREP110) is required for the amplification of the target replicon 11,14,16. Furthermore, the inclusion of a vector encoding the silencing suppressor p19 from tomato bushy stunt virus is desired for high level target gene expression 11,16.
There are generally three major steps for the introduction of genes of recombinant proteins into plant cells by agroinfiltration including plant growth, A. tumefaciens culture preparation, and infiltration. As every step is critical for the ultimate success of this procedure, therefore, a detailed description for each is provided for both syringe infiltration and vacuum infiltration below.

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents</td>
		 </tr>
		 <tr>
			<td>GFP </td>
			<td>Invitrogen</td>
			<td>V353-20</td>
			<td><a href="http://www.invitrogen.com/" target="_blank">www.invitrogen.com</a> See reference: Lico and Chen, et al 2008</td>
		 </tr>
		 <tr>
			<td>DsRed</td>
			<td>Clontech</td>
			<td>632152</td>
			<td><a href="http://www.clontech.com/" target="_blank">www.clontech.com</a> See reference: Baird and Zacharias, et al 2000</td>
		 </tr>
		 <tr>
			<td>MagnICON Vector</td>
			<td>Icon Genetics</td>
			<td>n/a</td>
			<td><a href="http://www.icongenetics.com/" target="_blank">www.icongenetics.com</a> See reference: giritch and Marillonnet, et al 2006</td>
		 </tr>
		 <tr>
			<td>Geminiviral Vector</td>
			<td>Author's Lab</td>
			<td>n/a</td>
			<td>See reference: Chen and He, et al 2011</td>
		 </tr>
		 <tr>
			<td>N. benthamiana</td>
			<td>Author's Lab</td>
			<td>n/a</td>
			<td><a href="http://www.herbalistics.com.au" target="_blank">herbalistics.com.au</a></td>
		 </tr>
		 <tr>
			<td>Agrobacterium tumefaciens strain gv3101</td>
			<td>Author's Lab</td>
			<td>n/a</td>
			<td>See reference: Lai and Chen 2012</td>
		 </tr>
		 <tr>
			<td>LB Agar Carbenicillin-100, plates</td>
			<td>Sigma</td>
			<td>L0418</td>
			<td><a href="http://www.sigmaaldrich.com/" target="_blank">www.sigmaaldrich.com</a></td>
		 </tr>
		 <tr>
			<td>LB Agar Kanamycin-50, plates</td>
			<td>Sigma</td>
			<td>L0543</td>
			<td><a href="http://www.sigmaaldrich.com/" target="_blank">www.sigmaaldrich.com</a></td>
		 </tr>
		 <tr>
			<td>Magnesium sulfate hepa hydrate</td>
			<td>Sigma</td>
			<td>M2773-500 g</td>
			<td><a href="http://www.sigmaaldrich.com/" target="_blank">www.sigmaaldrich.com</a></td>
		 </tr>
		 <tr>
			<td>Bacto-Tryptone</td>
			<td>Fisher</td>
			<td>73049-73-7</td>
			<td><a href="http://www.fishersci.com/" target="_blank">www.fishersci.com</a></td>
		 </tr>
		 <tr>
			<td>Bacto Yeast Extract</td>
			<td>Becton, Dickinson &amp; CO.</td>
			<td>REF 212750</td>
			<td><a href="http://www.bd.com/" target="_blank">www.bd.com</a></td>
		 </tr>
		 <tr>
			<td>Difco Nutrient Broth</td>
			<td>Becton, Dickinson &amp; CO.</td>
			<td>REF 234000</td>
			<td><a href="http://www.bd.com/" target="_blank">www.bd.com</a></td>
		 </tr>
		 <tr>
			<td>MES hydrate Buffer</td>
			<td>Sigma</td>
			<td>M8250-1kg</td>
			<td><a href="http://www.sigmaaldrich.com/" target="_blank">www.sigmaaldrich.com</a></td>
		 </tr>
		 <tr>
			<td>Carbenicillin</td>
			<td>Sigma</td>
			<td>C1613-1ML</td>
			<td><a href="http://www.sigmaaldrich.com/" target="_blank">www.sigmaaldrich.com</a></td>
		 </tr>
		 <tr>
			<td>Kanamycin</td>
			<td>Sigma</td>
			<td>70560-51-9</td>
			<td><a href="http://www.sigmaaldrich.com/" target="_blank">www.sigmaaldrich.com</a></td>
		 </tr>
		 <tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma</td>
			<td>221465</td>
			<td><a href="http://www.sigmaaldrich.com/" target="_blank">www.sigmaaldrich.com</a></td>
		 </tr>
		 <tr>
			<td>Jack's Fertilizer</td>
			<td>Hummert International</td>
			<td>Jul-25</td>
			<td><a href="http://www.hummert.com/" target="_blank">www.hummert.com</a></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		 </tr>
		 <tr>
			<td>Vacuubrand MD4 Vacuum Pump</td>
			<td>Fisher</td>
			<td>13-878-113</td>
			<td><a href="http://www.fishersci.com/" target="_blank">www.fishersci.com</a></td>
		 </tr>
		 <tr>
			<td>Vacuum Air Regulator Valve</td>
			<td>Fisher</td>
			<td>NC9386590</td>
			<td><a href="http://www.fishersci.com/" target="_blank">www.fishersci.com</a></td>
		 </tr>
		 <tr>
			<td>Desiccator 12 1/8" with O ring</td>
			<td>Fisher</td>
			<td>08-594-15C </td>
			<td><a href="http://www.fishersci.com/" target="_blank">www.fishersci.com</a></td>
		 </tr>
		 <tr>
			<td>3 L Tub</td>
			<td>Rubber-Maid</td>
			<td>n/a</td>
			<td>Rubbermaid Servn' Saver Bowl, 10-cup will work</td>
		 </tr>
		 <tr>
			<td>plate/shelf 230ML</td>
			<td>Fisher</td>
			<td>NC9489269</td>
			<td><a href="http://www.fishersci.com/" target="_blank">www.fishersci.com</a></td>
		 </tr>
		 <tr>
			<td>Peat Pellet</td>
			<td>Hummert International</td>
			<td>14-2370-1</td>
			<td><a href="http://www.hummert.com/" target="_blank">www.hummert.com</a></td>
		 </tr>
		 <tr>
			<td>Propagation Tray Dome</td>
			<td>hydrofarm</td>
			<td>132052</td>
			<td><a href="http://www.hydroponics.net/" target="_blank">www.hydroponics.net</a></td>
		 </tr>
		 <tr>
			<td>Propagaiton Tray</td>
			<td>hydrofarm</td>
			<td>138758</td>
			<td><a href="http://www.hydroponics.net/" target="_blank">www.hydroponics.net</a></td>
		 </tr>
		 <tr>
			<td>Virbo Hand Seeder</td>
			<td>Gro-Mor INC</td>
			<td>n/a</td>
			<td><a href="http://www.gro-morent.com/" target="_blank">www.gro-morent.com</a></td>
		 </tr>
		 <tr>
			<td>Flora Cart 4 shelf</td>
			<td>Hummert International</td>
			<td>65-6924-1</td>
			<td><a href="http://www.hummert.com/" target="_blank">www.hummert.com</a></td>
		 </tr>
		 <tr>
			<td>15 ml Round Bottom Culture Tubes</td>
			<td>Sigma</td>
			<td>CLS430172-500EA</td>
			<td><a href="http://www.sigmaaldrich.com/" target="_blank">http://www.sigmaaldrich.com</a></td>
		 </tr>
		 <tr>
			<td>Spectrophotometer</td>
			<td>Bio-Rad</td>
			<td>170-2525</td>
			<td><a href="http://www.bio-rad.com/" target="_blank">www.bio-rad.com</a></td>
		 </tr>
		 <tr>
			<td>Spectrophotometer Cuvettes</td>
			<td>Bio-Rad</td>
			<td>223-9950</td>
			<td><a href="http://www.bio-rad.com/" target="_blank">www.bio-rad.com</a></td>
		 </tr>
		 <tr>
			<td>Microcentrifuge Tubes</td>
			<td>USA Scientific</td>
			<td>1415-2500</td>
			<td><a href="http://www.usascientific.com/" target="_blank">www.usascientific.com</a></td>
		 </tr>
		 <tr>
			<td>Benchtop Centrifuge</td>
			<td>Bio-Rad</td>
			<td>166-0602EDU</td>
			<td><a href="http://www.bio-rad.com/" target="_blank">www.bio-rad.com</a></td>
		 </tr>
		 <tr>
			<td>Incubator/Shaker</td>
			<td>Eppendorf</td>
			<td>Excella E25</td>
			<td><a href="http://www.eppendorf.com/" target="_blank">www.eppendorf.com</a></td>
		 </tr>
		 <tr>
			<td>Ultraviolet Light Model#: UVGL-25</td>
			<td>UVP</td>
			<td>95-0021-12</td>
			<td><a href="http://www.uvp.com/" target="_blank">www.uvp.com</a></td>
		 </tr>
		</tbody>
 </table>

efficient agroinfiltration of plants for high-level transient expression of recombinant proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

1. Expression of Fluorescent Proteins by Syringe Infiltration
To demonstrate the effectiveness of syringe infiltration of Agrobacterium into plant tissue, we tested the expression of two fluorescent proteins - GFP and DsRed - by two different deconstructed plant viral vectors - geminiviral and MagnICON - in N. benthamiana. For N. benthamiana leaves that were entirely infiltrated with Agrobacteria containing geminiviral vectors, GFP expression was observed over the entire leaf area under UV light starting from as early as 2 dpi and reached peak accumulation at 4 dpi (Figure 4C). This early GFP expression by geminiviral vector is consistent with previous results of other recombinant proteins 14,16,18,19. In contrast, leaves infiltrated with MagnICON vector-containing Agrobacterium combinations showed GFP fluorescence only after 5 dpi and reached its maximum accumulation at 7 dpi (Figure 4D). No green fluorescence was observed from leaves infiltrated with negative control Agrobacterium mixtures of pREP110 + p19 (Figure 4B) or pICH15879 + pCH14011 (data not shown), indicating that the fluorescence was specific to the GFP gene and was not the result of background fluorescence from the leaves. At its peak accumulation, the fluorescence of MagnICON vector-expressed GFP is more intense than that of geminiviral vector (Figure 4C and 4D), similar to results previously obtained for other recombinant proteins 8,18. The temporal expression pattern and the relative fluorescent intensity of DsRed are similar to that of GFP (data not shown). No major necrosis was observed on leaves infiltrated with GFP constructs (Figure 4A). Similar results were also observed for DsRed-expressing plants, indicating that neither fluorescent protein is highly toxic to plant cells. However, local necrosis at the nick site was observed and they appeared as "holes" in the photographs (Figure 4). When both florescent proteins were expressed on the same leaf via geminiviral vectors with syringe spot-infiltration, they were detected with their expected fluorescent color in the spot where they were infiltrated (Figure 5). Interestingly, co-infiltration of GFP and DsRed resulted in yellowish fluorescence (Figure 5). The intensity of DsRed fluorescence appeared to be weaker than that of GFP on the same leaf. However, this may not reflect the weaker expression of DsRed, but rather due to the less-than-optimal excitation of DsRed by UV light. Overall, our results demonstrate that syringe infiltration efficiently introduces recombinant gene-carrying Agrobacteria into plant leaves and resulted in robust expression of our target proteins. In addition, we demonstrated that this method allows the flexibility to either infiltrate entire leaves for maximal accumulation of one target protein or to spot-infiltrate multiple protein targets with multiple vectors for comparing their expression and expression pattern.
2. Expression of Fluorescent Proteins by Vacuum Infiltration
Vacuum infiltration was also examined to develop a scalable agroinfiltration method that can be used for large-scale production of recombinant proteins by plant transient expression systems. Our results indicate that the temporal expression pattern of GFP or DsRed by geminiviral and MagnICON vectors was not altered by the change of infiltration method and remained similar to that of syringe infiltration. As the entire shoot of a plant is submerged in the infiltration mixture during vacuum infiltration, fluorescence was observed for GFP (Figure 6) for all leaves of the infiltrated plants. Compared with syringe infiltration, it is more robust and can achieve infiltration of each plant with a much shorter time frame. For example, it takes a skilled student 15 min to syringe-infiltrate one entire 6-week old N. benthamiana plant. In contrast, the same plant can be infiltrated in 3 min by vacuum from start to finish and multiple plants can be infiltrated at the same time.
<img alt="Figure 1" src="/files/ftp_upload/50521/50521fig1.jpg" /> 
 Figure 1. N. benthamiana plant growth with peat pellets and growth trays. Wild-type plants at 0 (A), 2 (B), 4 (C) and 6 (D) weeks after seeding.
<img alt="Figure 2" fo:content-width="6in" fo:src="/files/ftp_upload/50521/50521fig2highres.jpg" src="/files/ftp_upload/50521/50521fig2.jpg" /> 
 Figure 2. Syringe infiltration of N. benthamiana leaves with Agrobacterium tumefaciens. Strain GV3101 of A. tumefaciens harboring GFP-expressing MagnICON vectors were resuspended in infiltration buffer and loaded into a syringe without a needle. A nick was created with a needle on the back side of a 6-week old plant leaf (A). The opening of the syringe was positioned against the nick (B) and Agrobacteria in infiltration buffer were injected into the intercellular space of the leaf via the nick (C).
<img alt="Figure 3" fo:content-width="6in" fo:src="/files/ftp_upload/50521/50521fig3highres.jpg" src="/files/ftp_upload/50521/50521fig3.jpg" /> 
 Figure 3. Vacuum infiltration of N. benthamiana leaves with Agrobacterium tumefaciens. Strain GV3101 of A. tumefaciens containing target protein-expressing vectors were resuspended in infiltration buffer and loaded into a 3 L tub. The tub was then placed into a vacuum desiccator that was connected to a vacuum pump (A). A 6-week old plant was placed upside down on the desiccator plate (B). The entire leaf and stem system was then submerged into the tub as the plate was placed atop the chamber (C). Agroinfiltration was achieved by applying and releasing a vacuum at 100 mbar twice for 1 min. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50521/50521fig3large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 4" src="/files/ftp_upload/50521/50521fig4.jpg" /> 
 Figure 4. Expression of GFP in syringe agroinfiltrated leaves. Entire N. benthamiana leaves were infiltrated with strain GV3101 of A. tumefaciens harboring the GFP gene in a geminiviral (A and C) or MagnICON vector (D). Negative control leaves were infiltrated with Agrobacteria strains that do not contain that GFP gene (B). Leaves were photographed under white light (A) or UV light (B-D) at 4 dpi (A-C) or 7 dpi (D).
<img alt="Figure 5" src="/files/ftp_upload/50521/50521fig5.jpg" /> 
 Figure 5. Expression of GFP and DsRed in syringe spot-agroinfiltrated leaves. N. benthamiana leaves were spot-infiltrated with GV3101 harboring the GFP, DsRed, or both GFP and DsRed genes in geminiviral vectors. A negative control spot was infiltrated with pREP110+p19. Leaves were photographed under UV light at 4 dpi.
<img alt="Figure 6" src="/files/ftp_upload/50521/50521fig6.jpg" /> 
 Figure 6. Expression of GFP in vacuum agroinfiltrated leaves. N. benthamiana leaves were infiltrated with GV3101 harboring the GFP gene in MagnICON vectors. Leaves were photographed under UV light at 7 dpi.

Watch this Scientific Journal Video about Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins at JoVE.com

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

efficient-agroinfiltration-plants-for-high-level-transient-expression

Nanyang Technological University

Research

JoVE Journal

Biology

48.5K Views.  Arizona State University. Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

Video: Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

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 ...

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and easy to use expression system.
As a single-celled microorganism P. pastoris is easy to manipulate and grows rapidly on inexpensive media at high cell densities. Being a eukaryote, P. pastoris is able to perform many of the post-translational modifications performed by higher eukaryotic cells and the obtained recombinant proteins undergo protein folding, proteolytic processing, disulfide bond formation and glycosylation [1].
As a methylotrophic yeast P. pastoris is capable of metabolizing methanol as its sole carbon source. The strong promoter for alcohol oxidase, AOX1, is tightly regulated and induced by methanol and it is used for the expression of the gene of interest. Accordingly, the expression of the foreign protein can be induced by adding methanol to the growth medium [2; 3].
Another important advantage is the secretion of the recombinant protein into the growth medium, using a signal sequence to target the foreign protein to the secretory pathway of P. pastoris. With only low levels of endogenous protein secreted to the media by the yeast itself and no added proteins to the media, a heterologous protein builds the majority of the total protein in the medium and facilitates following protein purification steps [3; 4].
The vector used here (pPICZ&alpha;A) contains the AOX1 promoter for tightly regulated, methanol-induced expression of the gene of interest; the &alpha;-factor secretion signal for secretion of the recombinant protein, a Zeocin resistance gene for selection in both E. coli and Pichia and a C-terminal peptide containing the c-myc epitope and a polyhistidine (6xHis) tag for detection and purification of a recombinant protein. We also show western blot analysis of the recombinant protein using the specific Anti-myc-HRP antibody recognizing the c-myc epitope on the parent vector.

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

The protocol describes protein expression using the methylotrophic yeast Pichia pastoris. The preparation of electrocompetent yeast cells, transformation of the vector with the gene of interest into P. pastoris and yeast DNA purification are also performed. Western blot analysis and protein purification build the last steps in this protein expression protocol.

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4&#160;ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (<a href="http://www.venomics.eu">www.venomics.eu</a>), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past years, hydrodynamic gene delivery (HGD) has emerged as a simple, fast, safe and effective method for delivering transgenes into rodents. This technique relies on the force generated by the rapid injection of a large volume of physiological solution to increase the permeability of cell membranes of perfused organs and thus deliver DNA into cells. One of the main advantages of HGD is the ability to introduce transgenes into mammalian cells using naked plasmid DNA (pDNA). Introducing an exogenous gene using a plasmid is minimally laborious, highly efficient and, contrary to viral carriers, remarkably safe. HGD was initially used to deliver genes into mice, it is now used to deliver a wide range of substances, including oligonucleotides, artificial chromosomes, RNA, proteins and small molecules into mice, rats and, to a limited degree, other animals. This protocol describes HGD in mice and focuses on three key aspects of the method that are critical to performing the procedure successfully: correct insertion of the needle into the vein, the volume of injection and the speed of delivery. Examples are given to show the application of this method to the transient expression of two genes that encode secreted, primate-specific proteins, apolipoprotein L-I (APOL-I) and haptoglobin-related protein (HPR).

In vivo transfection of naked DNA by hydrodynamic gene delivery introduces genes into the tissue of an animal with minimal inflammatory response. Sufficient amounts of gene product are generated such that gene function and regulation as well as protein structure and function can be analyzed.

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past ...

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is the challenge of identifying suitable vaccine candidates. Malarial proteins expressed using prokaryotic and eukaryotic cell based expression systems are poorly glycosylated, generally insoluble and undergo improper folding leading to reduced immunogenicity. The wheat germ, rabbit reticulocyte lysate and Escherichia coli lysate cell free expression systems are currently used for expression of malarial proteins. However, the length of expression time and improper glycosylation of proteins still remains a challenge. We demonstrate expression of Plasmodium proteins in vitro using HeLa based cell free expression systems, termed &#8220;in vitro human cell free expression systems&#8221;. The 2 HeLa based cell free expression systems transcribe mRNA in 75 min and 3 &#181;l of transcribed mRNA is sufficient to translate proteins in 90 min. The 1-step expression system is a transcription and translation coupled expression system; the transcription and co-translation occurs in 3 hr. The process can also be extended for 6 hr by providing additional energy. In the 2-step expression system, mRNA is first transcribed and then added to the translation mix for protein expression. We describe how to express malaria proteins; a hydrophobic PF3D7_0114100 Maurer&#8217;s Cleft &#8211; 2 transmembrane (PfMC-2TM) protein, a hydrophilic PF3D7_0925900 protein and an armadillo repeats containing protein PF3D7_1361800, using the HeLa based cell free expression system. The proteins are expressed in micro volumes employing 2-step and 1-step expression strategies. An affinity purification method to purify 25 &#181;l of proteins expressed using the in vitro human cell free expression system is also described. Protein yield is determined by Bradford&#8217;s assay and the expressed and purified proteins can be confirmed by western blotting analysis. Expressed recombinant proteins can be used for immunizations, immunoassays and protein sequencing.

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

Expression of malarial proteins in cell based systems remains challenging. We demonstrate two step and one step IVT (in vitro translation) cell free expression systems for expressing malarial recombinant rhoptry proteins from HeLa cells. We use a Ni-resin affinity based purification system to purify the rhoptry proteins.

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is ...

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time and cost efficient protocol for secreted protein expression in mammalian cells and one step purification using nickel affinity chromatography. The system is based on large scale transient transfection of mammalian cells in suspension, which greatly decreases the time to produce protein, as it eliminates steps, such as developing expression viruses or generating stable expressing cell lines. This protocol utilizes cheap transfection agents, which can be easily made by simple chemical modification, or moderately priced transfection agents, which increase yield through increased transfection efficiency and decreased cytotoxicity. Careful monitoring and maintaining of media glucose levels increases protein yield. Controlling the maturation of native glycans at the expression step increases the final yield of properly folded and functional mammalian proteins, which are ideal properties to pursue X-ray crystallography. In some cases, single step purification produces protein of sufficient purity for crystallization, which is demonstrated here as an example case.

This is a quick, cost-efficient protocol for the production of secreted, glycosylated mammalian proteins and subsequent single-step purification with sufficient yields of homogenous protein for X-ray crystallography and other biophysical studies.

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time ...

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human Fc&#947;RIIIa and the rat &#945;2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...