We would like to acknowledge Dr. Thomas Rademacher, Alexander Boes and Veronique Bei&#223; for providing the transgenic tobacco seeds, and Ibrahim Al Amedi for cultivating the tobacco plants. The authors wish to thank Dr. Richard M. Twyman for editorial assistance as well as G&#252;ven Edg&#252; for providing the MSP1-19 reference. This work was funded in part by the European Research Council Advanced Grant ''Future-Pharma'', proposal number 269110, the Fraunhofer-Zukunftsstiftung (Fraunhofer Future Foundation) and Fraunhofer-Gesellschaft Internal Programs under Grant No. Attract 125-600164.

1. Cultivate the Tobacco Plants
<ol>
	<li>Flush each mineral wool block with 1 to 2 L of deionized water and subsequently with 1 L of 0.1% [w/v] fertilizer solution. Place one tobacco seed in each mineral wool block and gently flush with 0.25 L of fertilizer solution without washing away the seed 16.</li>
	<li>Cultivate the tobacco plants for 7 weeks in a greenhouse with 70% relative humidity, a 16 hr photoperiod (180 &#181;mol sec&#8722;1 m&#8722;2;&#160;&#955; = 400 - 700 nm) and a 25/22 &#176;C light/dark temperature regime.</li>
	<li>Harvest all leaves except the four cotyledon leaves, which are located at the base of the plant stem.</li>
</ol>
2. Optional: Heat Precipitation by Blanching
NOTE: Carry out the steps described in steps 2.1 to 2.12 in order to precipitate tobacco HCPs by blanching. Skip the entire section 2, if the HCPs will be precipitated in a heated vessel (section 4) or using a heat exchanger (section 5).
<ol>
	<li>Set aside 50 g of plant material and carry out extraction without blanching (section 3). Take a sample of this extract as an internal control during subsequent analysis (section 7).</li>
	<li>Set up an 8-L working volume water bath, e.g., 50 x 40 x 40 cm, in a thermally-insulated polystyrene foam bucket or similar. Mount the adjustable thermostat on the water bath for subsequent temperature control (step 2.7).</li>
	<li>Transfer the entire assembly onto a magnetic stir plate and place a magnetic stir bar in the water bath. Ensure that the magnetic field is strong enough to rotate the stir bar.</li>
	<li>Surround the stir bar with at least four support tiles upon which a polypropylene basket (step 2.6) will be placed later during the blanching procedure. Make sure that the support tiles are made from a non-magnetic material (e.g., stainless steel alloy containing nickel) and that they are higher than the stir bar (Figure 2).</li>
	<li>Add 8 L of deionized water to the water bath. 
	NOTE: Using a buffer, e.g., 50 mM sodium phosphate (pH 7.5), can improve the target protein yields if low pH precipitation is an issue.</li>
	<li>Place a 23 x 23 x 23 cm polypropylene basket on the support tiles and make sure that the basket does not interfere with the stir bar rotation and that it is fully submerged in the liquid. If necessary, add additional water/buffer until the basket is fully submerged, then remove the basket again. 
	CAUTION: All subsequent steps up to 2.12 involve the handling of hot liquid. Wear appropriate personal protective equipment including thermally insulated gloves.</li>
	<li>Use the adjustable thermostat to bring the water bath to 70 &#176;C (or the temperature required for the experiments). Wait at least 15 min after the desired temperature is reached to ensure the entire assembly has reached a thermal equilibrium.</li>
	<li>Prepare 150 g aliquots from the harvested tobacco leaves. Place one aliquot in the basket while avoiding irreversible compression and damage to the leaves, e.g., by tearing. Avoid overfilling the basket with plant material or a dense packing of the latter.</li>
	<li>Carefully but quickly submerge the basket in the hot liquid and place it on the support tiles. Place a stainless steel block on top of the basket to prevent flotation.</li>
	<li>Incubate the leaves for 5 min in the blanching fluid, or select a time suiting the experimental design. Monitor the liquid temperature during the entire incubation period.</li>
	<li>Carefully remove the basket from the blanching fluid and let residual liquid drain from the leaves for 30 sec. Then take the plant leaves out of the basket, transfer them to the blender and immediately start the extraction (section 3). 
	NOTE: Plants can be kept for extended periods of time after blanching and before the start of the extraction, e.g., more than 30 min on ice or frozen at -20 &#176;C for several weeks has been successfully tested. However, product stability may decline with increasing storage times and thus immediate processing is recommended.</li>
	<li>Repeat steps 2.8 to 2.11 with fresh leaf material until the entire harvested biomass is processed.</li>
</ol>
3. Protein Extraction from Tobacco Leaves
CAUTION: The next steps involve a blender with rotating blades. Do not work in the blender bucket while it is mounted on the blender motor.
<ol>
	<li>Place 150 g (wet mass) of harvested (step 1.3) or blanched (step 2.11) leaves in the blender and add 450 ml of extraction buffer (50 mM sodium phosphate, 500 mM sodium chloride, 10 mM sodium disulfite, pH 8.0). 
	NOTE: The extraction buffer composition depends on the protein to be extracted and can thus require adjustment, e.g., use of another pH or buffer component such as Tris.</li>
	<li>Homogenize the leaves for 3 x 30 sec pulses with 30 sec interspersed breaks. Ensure that the leaves are homogenized and do not clog the blender bucket. Stop the blender and lift the leaves to prevent clogging if necessary, and then continue the homogenization.</li>
	<li>Take a 1 ml sample of each extract that is produced for subsequent analysis (section 7). If the plant material is blanched, continue to section 6. Otherwise continue with heat precipitation in a vessel (section 4) or heat exchanger (section 5), depending on the selected experimental approach.</li>
</ol>
4. Optional: Heat Precipitation in a Stirred Vessel
NOTE: Conduct the steps described in sections 4.2 to 4.11 in order to precipitate tobacco HCPs in a stirred vessel. Skip the entire section 4, if HCPs have been precipitated by blanching (section 2) or will be precipitated using a heat exchanger (section 5).
<ol>
	<li>Set up two 8-L working volume water baths, e.g., 50 x 40 x 40 cm, in thermally insulated polystyrene foam or similar. In the first bath, mount the adjustable thermostat for subsequent temperature control (step 4.6). In the second, add 5 L of deionized water and 2 kg of ice for subsequent cooling (step 4.9).</li>
	<li>Transfer the first water bath onto a magnetic stir plate, and place the 2-L stainless steel vessel into the water bath such that the center of the vessel is aligned to the center of the stir plate.</li>
	<li>Place a magnetic stir bar in the stainless-steel vessel. Ensure that the magnetic field is strong enough to rotate the stir bar.</li>
	<li>Fill the water bath with deionized water to 5 cm below the upper edge of the stainless steel vessel. Then fill the vessel with extraction buffer. Place a polystyrene foam lid on the vessel. 
	CAUTION: All subsequent steps up to 4.9 involve the handling of hot liquid. Wear appropriate personal protective equipment including thermally insulated gloves.</li>
	<li>Insert a thermometer into the stainless steel vessel through a suiting hole in the polystyrene foam lid. Set the water bath temperature to 78 &#176;C and incubate the entire assembly for 15 min to reach thermal equilibrium. Ensure that the temperature in the vessel is 70 &#176;C, approximately 8 &#176;C below the set point of the water bath.</li>
	<li>If the temperature in the stainless steel vessel differs from 70 &#176;C, adjust the temperature of the water bath accordingly and let the system equilibrate for another 15 min. Repeat this step until the temperature in the vessel is 70 &#176;C or as required for the experiment, and empty the stainless steel vessel.</li>
	<li>Pour 300 ml of extract (step 3.2) into the stainless-steel vessel while it is still in the water bath and start a timer. Stir the extract at 150 rpm and incubate for 5 min. Ensure that the extract reaches a temperature of 70 &#176;C for at least 2 min during this incubation period.</li>
	<li>Remove the hot water bath from the magnetic stirrer, take out the stainless steel vessel and place it in the ice-cold bucket. Place the latter onto the magnetic stirrer and remove the polystyrene foam lid.</li>
	<li>Ensure that the plant homogenate is well agitated at 150 rpm, place the thermometer in the extract and incubate until it reaches a temperature of 20 &#176;C or the temperature specified by the experimental design.</li>
	<li>Repeat steps 4.7 to 4.9 for all aliquots. Take a 1 ml sample of each heat precipitated extract once it has reached the final temperature, then proceed with section 6.</li>
</ol>
5. Optional: Heat Precipitation in a Heat Exchanger
NOTE: Conduct the steps described in sections 5.2 to 5.12 in order to precipitate tobacco HCPs using a heat exchanger. Skip the entire section 5, if HCPs have been precipitated by blanching (section 2) or in a heated vessel (section 4).
<ol>
	<li>Set up two 8-L working volume water baths, e.g., 50 x 40 x 40 cm, in thermally-insulated polystyrene foam buckets or similar. In the first bath, mount the adjustable thermostat for subsequent temperature control (step 5.3). In the second, add 5 L of deionized water and 2 kg of ice for subsequent cooling (step 5.10). 
	CAUTION: All subsequent steps up to 5.9 involve the handling of hot liquid. Wear appropriate personal protective equipment including thermally insulated gloves.</li>
	<li>Fill the first water bath with 8 L deionized water. Set the temperature to 74.5 &#176;C using the thermostat. Incubate the assembly for 15 min to reach thermal equilibrium.</li>
	<li>Prepare an insulated storage vessel by placing a 0.5-L plastic beaker into a 1-L plastic beaker and fill the gaps with cotton wool. Alternatively, use a dedicated thermally insulated vessel with a 0.5-L working volume.</li>
	<li>Connect the heat exchanger to the peristaltic pump at one end and to an outlet hose on the other end using L/S 24 tubing. Place both tubing ends along with a thermometer in the insulated storage vessel and fill it with 300 ml extraction buffer (Figure 2).</li>
	<li>Place the heat exchanger into the hot water bath and start the peristaltic pump at a rate of 300 ml min-1. Ensure that the resulting temperature in the vessel is 70 &#176;C, approximately 4.5 &#176;C below the set point of the water bath, after 3 min.</li>
	<li>If the temperature in the insulated-vessel differs from 70 &#176;C, adjust the temperature of the water bath accordingly and let the system equilibrate for another 15 min. Repeat this step until the temperature of the extraction buffer increases from ambient to 70 &#176;C in less than 3 min, or equals that required for the experiment, and empty the stainless-steel vessel.</li>
	<li>Discard the extraction buffer from the insulated vessel and prepare 300-ml aliquots of the plant extract. Fill the insulated vessel with one aliquot.</li>
	<li>Pump the plant extract (section 3.3) through the heat exchanger at 300 ml min-1 for 5 min. Ensure that the extract temperature is 70 &#176;C after 3 min, or equals the temperature defined in the experimental design.</li>
	<li>After 5 min, place the heat exchanger into the ice-cold water bath while the extract is still being pumped. Incubate in this setting until the temperature reaches exactly 20 &#176;C, or the temperature defined in the experimental design.</li>
	<li>Remove the inlet hose connected to the peristaltic pump from the insulated vessel and continue pumping to collect residual heat-precipitated plant extract from within the heat exchanger. Then stop the pump.</li>
	<li>Repeat steps 5.7 to 5.10 for all aliquots. Take a 1-ml sample of each heat-precipitated extract once it has reached the final temperature, then pass on the extracts to bag filtration (section 6). 
	NOTE: After a first cycle through steps 5.7 to 5.10 there will be no extraction buffer to discard in step 5.7.</li>
</ol>
6. Bag Filtration of the Plant Extract
<ol>
	<li>Mount a bag filter into the corresponding support basket which is fitted into the filter housing and provides mechanical support for the flexible filter material. Place a 1-L vessel beneath the basket and apply the extract aliquots (section 3.3, 4.10 or 5.11 depending on the selected heat treatment) to the bag at a rate of 150 ml min-1.</li>
	<li>After filtration, measure the turbidity of a 1:10 dilution of filtrate in extraction buffer using the turbidimeter.</li>
	<li>Take a 1 ml sample and process the bag filtrate as defined in the experimental design, e.g., depth filtration and chromatography 7,10.</li>
</ol>
7. Sample Analysis
<ol>
	<li>Measure the quantity of total soluble protein (TSP) using the Bradford method 24,25.
	<ol>
		<li>In triplicate, pipette 2.5 &#181;l of each sample into the single wells of a 96-well plate. Include eight bovine serum albumin (BSA) standards in triplicate covering the range 0 - 2,000 &#181;g ml-1.</li>
		<li>Add 200 &#181;l Bradford reagent to each well and mix thoroughly by pipetting up and down but gently enough to avoid forming bubbles.</li>
		<li>Incubate for 10 min at 22 &#176;C and measure the absorbance at 595 nm in a spectrophotometer. Calculate the TSP concentration in the samples based on a standard curve through the BSA reference points.</li>
	</ol>
	</li>
	<li>Quantify Vax8 by surface plasmon resonance spectroscopy 26.
	<ol>
		<li>Setup the surface plasmon resonance instrument with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) running buffer (10 mM HEPES, 3 mM ethylenediaminetetraacetic acid (EDTA), 1,500 mM sodium chloride, 0.05% v/v polysorbate 20, pH 7.4) and mount a carboxymethylated dextran surface chip into the device. Use the prime function to flush the system and start a manual run with a flow rate of 30 &#181;l min-1 over flow cells 1 and 2. Inject 30 mM hydrogen chloride twice for 60 sec with a 60 sec interspersed injection of 25 mM sodium hydroxide.</li>
		<li>Prepare 200 &#181;l of a 500 &#181;g ml-1 mAb 5.2 solution in 10 mM sodium acetate (pH 4.0). Thaw 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) vials and centrifuge them at 16,000 x g for 1 min. Mix 70 &#181;l of EDC with 70 &#181;l of NHS and transfer to a 7-mm plastic vial.</li>
		<li>Activate the carboxymethylated dextran surface chip surface by injecting the EDC/NHS mixture over flow cells 1 and 2 for 10 min with a flow rate of 10 &#181;l min-1.</li>
		<li>Set the flow path to flow cell 2 only. Couple mAb 5.2 by injecting it for 15 min at a rate of 10 &#181;l min-1.</li>
		<li>Switch the flow path to flow cells 1 and 2 and inject ethanolamine for 7 min at a rate of 5 &#181;l min-1 to deactivate the surface. Then inject 30 mM hydrogen chloride twice for 60 sec with a 60 sec interspersed injection of 25 mM sodium hydroxide.</li>
		<li>Inject the samples and MSP1 - 19 standards at a rate of 30 &#181;l min-1 for 180 sec. Ensure that the Vax8 concentration is 50 - 1,000 ng ml-1. Prepare pre-dilutions in HEPES buffered saline containing EDTA and polysorbate 20 (HBSEP) if necessary.</li>
		<li>Subtract the peak signal of flow cell 1 from that of flow cell 2 and use the difference to calculate the Vax8 concentration in samples based on the signal of the MSP 1 - 19 injection with a known protein concentration of 500 ng ml-1.</li>
		<li>Regenerate the chip surface after each sample or MSP 1 - 19 injection by exposure to 30 mM hydrogen chloride for 60 sec at a flow rate of 30 &#181;l min-1.</li>
	</ol>
	</li>
	<li>Quantify DsRed by fluorescence spectrometry
	<ol>
		<li>In triplicate, pipette 50 &#181;l of each sample into the single wells of a black half-area 96-well plate. Include six DsRed standards covering the range 0 - 225 &#181;g ml-1.</li>
		<li>Measure the fluorescence in duplicate using a 530 &#177; 30 nm excitation filter and a 590 &#177; 35 nm emission filter in a spectrophotometer. Calculate the DsRed concentration in the samples based on a standard curve through the DsRed reference points.</li>
	</ol>
	</li>
	<li>Determination of particle size distributions
	<ol>
		<li>Wash a cuvette with 1 ml isopropanol and then with 2 ml of deionized water to remove dust particles. Pipette 850 &#181;l of the sample into the cuvette.</li>
		<li>Place the cuvette into the zeta potential and particle size analyzer. Open the &#34;Software&#34; and start a manual measurement with &#34;Protein&#34; as the material and &#34;PBS&#34; as the dispersant. Select a temperature of 25 &#176;C and an equilibration time of 180 sec.</li>
		<li>Choose the &#34;DTS0012&#34; cell and measure at 173&#176; backscatter. Check the &#34;auto&#34; function for the measurement duration and select three measurements with no interspersed delay.</li>
		<li>Investigate the particle size distribution peak profile once the measurement is complete by selecting the three measurements of the sample in the experiment view. Choose the &#34;Volume PSD&#34; tab.</li>
	</ol>
	</li>
</ol>

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The authors have no conflicts of interest to disclose.

The three methods for heat precipitation described above can effectively remove tobacco HCPs prior to any chromatographic purification step 16,17. They complement other strategies that aim to increase initial product purity, e.g., guttation 29, rhizosecretion 30 or centrifugal extraction 31,32, all of which are limited to secreted proteins. However, the heat-based methods can only be used in a meaningful way if the target protein to be purified can withstand the minimu...

Modern healthcare systems increasingly depend on biopharmaceutical proteins 1. Producing these proteins in plants is advantageous due to the low pathogen burden and greater scalability compared to conventional expression systems 2-4. However, the downstream processing (DSP) of plant-derived pharmaceuticals can be challenging because the disruptive extraction procedures result in a high particle burden, with turbidities exceeding 5,000 nephelometric turbidity units (NTUs), and host cell protein (HCP) concentrations often exceeding 95% [m/m] 5,6.
Elaborate clarification procedures are required to remove dispersed particles 7-9, but chromatography equipment is less expensive to operate in bind-and-elute mode during initial product recovery if there is an earlier step for the efficient removal of HCPs 10,11. This can be achieved either by precipitating the target protein using flocculants 12 or low pH 13,14, as well as by causing the HCPs to aggregate. The selective aggregation of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the most abundant HCP in green plants such as tobacco (Nicotiana tabacum), can be promoted by adding polyethylene glycol 15, but this is expensive and incompatible with large-scale manufacturing. Heat treatment has been shown to denature and precipitate more than 95% of tobacco HCPs, while protein malaria vaccine candidates such as Vax8 remain stable in solution 16-18.
Three different approaches were used to achieve the heat-induced precipitation of tobacco HCPs: (i) blanching, i.e., the immersion of intact leaves in hot liquid, (ii) a temperature-controlled stirred vessel, and (iii) a heat exchanger (Figure 1) 16. For intact leaves, blanching achieved the rapid and efficient precipitation of HCPs and was also easy to scale up and compatible with existing large-scale manufacturing processes that include an initial step to wash the plant biomass 19. In contrast, temperature-controlled vessels are already available in some processes and can be used for the thermal treatment of plant extracts 20, but their scalability and energy transfer rate are limited because the surface-to-volume ratio of the tanks is progressively reduced and becomes unsuitable at process scale. A heat exchanger is a technically well-defined alternative to heated stirred vessels but requires an abundant supply of heating and cooling media, e.g., steam and cold water, as well as a tightly controlled volumetric flow rate that is adapted to the heat exchanger geometry and media properties, e.g., the specific heat capacity. This article shows how all three methods can be used for the heat-induced precipitation of tobacco HCPs, and plant HCPs in general. The establishment and operation of each method in a laboratory setting can be used to evaluate their suitability for larger-scale processes. The major challenge is to identify adequate scale-down models and running conditions for each operation that resemble the devices and conditions used during process-scale manufacturing. The data presented here refer to experiments conducted with transgenic tobacco plants expressing the malaria vaccine candidate Vax8 and fluorescent protein DsRed 16, but the method has also been successfully applied to N. benthamiana plants transiently expressing other biopharmaceutical proteins 21.
A design-of-experiments (DoE) approach 22 can facilitate process development, and flocculants 23 can also be beneficial in this context as previously described 8. The main difference between blanching, heated vessels and heat exchangers is that blanching is applied to intact leaves early in the process whereas the others are applied to plant extracts (Figure 1).
<img alt="Figure 1" src="/files/ftp_upload/54343/54343fig1.jpg" /> 
Figure 1: Process Flow Scheme Illustrating the Implementation of Three Different Methods for Tobacco HCP Heat Precipitation. The plant material is washed and homogenized before clarification and purification. The equipment for the blanching step (red) can easily be added to the existing machinery. In contrast, using a stirred vessel (orange) and especially a heat exchanger (blue) requires one or several additional devices and tubing. <a href="https://www.jove.com/files/ftp_upload/54343/54343fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="0" cellpadding="0" cellspacing="0" width="1026">
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			<td style="width:195px;">Eppendorf</td>
			<td style="width:119px;">5424 000.410</td>
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			<td style="width:119px;">2017106</td>
			<td>Reaction tube</td>
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			<td height="21" style="height:21px;width:360px;">Centrifuge tube 50 mL self-standing</td>
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			<td style="width:195px;">GE Healthcare</td>
			<td style="width:119px;">BR100012&#160;</td>
			<td>Chip for SPR measurements</td>
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			<td style="width:195px;">Stat-Ease, Inc.</td>
			<td style="width:119px;">n.a.</td>
			<td>DoE software</td>
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			<td height="21" style="height:21px;width:360px;">Disodium phosphate</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">&#160;4984.3&#160;</td>
			<td>Media component</td>
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			<td height="21" style="height:21px;width:360px;">Ferty 2 Mega</td>
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			<td style="width:119px;">88400</td>
			<td>Freezer</td>
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			<td height="21" style="height:21px;width:360px;">Greenhouse</td>
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			<td style="width:119px;">n.a.</td>
			<td>For plant cultivation</td>
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			<td height="21" style="height:21px;width:360px;">Grodan Rockwool Cubes 10x10cm</td>
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			<td>Rockwool block</td>
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			<td height="21" style="height:21px;width:360px;">Twentey-loop heat exchanger (4.8 m length)</td>
			<td style="width:195px;">n.a. (custom design)</td>
			<td style="width:119px;">n.a.</td>
			<td>Heat exchanger</td>
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			<td height="21" style="height:21px;width:360px;">HEPES</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">9105.3</td>
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			<td height="21" style="height:21px;width:360px;">K200P 60D</td>
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			<td height="21" style="height:21px;width:360px;">KS50P 60D</td>
			<td style="width:195px;">Pall</td>
			<td style="width:119px;">B12486</td>
			<td>Depth filter layer</td>
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			<td height="21" style="height:21px;width:360px;">Lauda E300</td>
			<td style="width:195px;">Lauda Dr Wobser GmbH</td>
			<td style="width:119px;">Z90010</td>
			<td>Water bath thermostat</td>
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			<td height="21" style="height:21px;width:360px;">L/S 24</td>
			<td style="width:195px;">Masterflex</td>
			<td style="width:119px;">SN-06508-24</td>
			<td>Tubing</td>
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			<td height="42" style="height:42px;width:360px;">mAb 5.2</td>
			<td style="width:195px;">American Type Culture Collection</td>
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			<td height="21" style="height:21px;width:360px;">Masterflex L/S</td>
			<td style="width:195px;">Masterflex</td>
			<td style="width:119px;">HV-77921-75</td>
			<td>Peristaltic pump</td>
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			<td height="21" style="height:21px;width:360px;">Miracloth</td>
			<td style="width:195px;">Labomedic</td>
			<td style="width:119px;">475855-1R</td>
			<td>Filter cloth</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:360px;">MultiLine Multi 3410 IDS</td>
			<td style="width:195px;">WTW</td>
			<td style="width:119px;">WTW_2020</td>
			<td>pH meter / conductivity meter</td>
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			<td height="21" style="height:21px;width:360px;">Osram cool white 36 W</td>
			<td style="width:195px;">Osram</td>
			<td style="width:119px;">4930440</td>
			<td>Light source</td>
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			<td height="21" style="height:21px;width:360px;">Phytotron</td>
			<td style="width:195px;">Ilka Zell</td>
			<td style="width:119px;">n.a.</td>
			<td>For plant cultivation</td>
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			<td height="21" style="height:21px;width:360px;">Sodium disulfit</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">8554.1</td>
			<td>Media component</td>
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			<td height="21" style="height:21px;width:360px;">Sodium chloride</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">P029.2</td>
			<td>Media component</td>
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			<td height="42" style="height:42px;width:360px;">Stainless-steel vessel; 0.7-kg 2.0-L; height 180 mm; diameter 120 mm</td>
			<td style="width:195px;">n.a. (custom design)</td>
			<td style="width:119px;">n.a.</td>
			<td>Container for heat precipitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Synergy HT</td>
			<td style="width:195px;">BioTek</td>
			<td style="width:119px;">SIAFRT</td>
			<td>Fluorescence and spectrometric plate reader</td>
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			<td height="21" style="height:21px;width:360px;">VelaPad 60</td>
			<td style="width:195px;">Pall</td>
			<td style="width:119px;">VP60G03KNH4</td>
			<td>Filter housing</td>
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			<td height="21" style="height:21px;width:360px;">Zetasizer Nano ZS</td>
			<td style="width:195px;">Malvern</td>
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			<td>DLS particle size distribution measurement</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Zetasizer Software v7.11</td>
			<td style="width:195px;">Malvern</td>
			<td style="width:119px;">n.a.</td>
			<td>Software to operate the Zetasizer Nano ZS device</td>
		</tr>
	</tbody>
</table>

comparison of tobacco host cell protein removal methods by blanching intact plants or by heat treatment of extracts

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical proteins, such as antibodies, vaccine candidates and enzymes. These must be purified from the plant biomass but chromatography steps are hindered by the high concentrations of host cell proteins (HCPs) in plant extracts. However, most HCPs irreversibly aggregate at temperatures above 60 &#176;C facilitating subsequent purification of the target protein. Here, three methods are presented to achieve the heat precipitation of tobacco HCPs in either intact leaves or extracts. The blanching of intact leaves can easily be incorporated into existing processes but may have a negative impact on subsequent filtration steps. The opposite is true for heat precipitation of leaf extracts in a stirred vessel, which can improve the performance of downstream operations albeit with major changes in process equipment design, such as homogenizer geometry. Finally, a heat exchanger setup is well characterized in terms of heat transfer conditions and easy to scale, but cleaning can be difficult and there may be a negative impact on filter capacity. The design-of-experiments approach can be used to identify the most relevant process parameters affecting HCP removal and product recovery. This facilitates the application of each method in other expression platforms and the identification of the most suitable method for a given purification strategy.

Heat precipitation of tobacco host cell proteins by blanching 
The blanching procedure described in section 2. was successfully used to precipitate HCPs from tobacco leaves with 70 &#176;C, reducing the TSP by 96 &#177; 1% (n = 3) while recovering up to 51% of the Vax8 target protein, thus increasing its purity from 0.1% to 1.2% before chromatographic separation 16. It was also possible to recover 83 &#177; 1% (n =3) of the fluorescent protein DsRed, increasing it...

Watch this Scientific Journal Video about Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts at JoVE.com

Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts

Three heat precipitation methods are presented that effectively remove more than 90% of host cell proteins (HCPs) from tobacco extracts prior to any other purification step. The plant HCPs irreversibly aggregate at temperatures above 60 &#176;C.

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical ...

comparison-tobacco-host-cell-protein-removal-methods-blanching-intact

Research

JoVE Journal

Biology

9.0K Views.  Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e. V.. Three heat precipitation methods are presented that effectively remove more than 90% of host cell proteins (HCPs) from tobacco extracts prior to any other purification step. The plant HCPs irreversibly aggregate at temperatures above 60 °C.

Video: Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 base pairs of DNA associated with two each of histones H2A, H2B, H3, and H41. The N-terminal tails of histones are rich in lysine and arginine and are modified post-transcriptionally by acetylation, methylation, and other post-translational modifications (PTMs). The PTM configuration of nucleosomes can affect the transcriptional activity of associated DNA, thus providing a mode of gene regulation that is epigenetic in nature 2,3. We developed a method called nucleosome ELISA (NU-ELISA) to quantitatively determine global PTM signatures of nucleosomes extracted from cells. NU-ELISA is more sensitive and quantitative than western blotting, and is useful to interrogate the epiproteomic state of specific cell types. This video journal article shows detailed procedures to perform NU-ELISA analysis.

Nucleosome ELISA (NU-ELISA) is a sensitive and quantitative method to detect global patterns of post-translational modifications in preparations of native, intact nucleosomes. These modifications include methylations, acetylations, and phosphorylations at specific histone amino acid residues, and hence NU-ELISA provides a global proteomic assay of the overall chromatin modification states of specific cell types.

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Generation of Transgenic Hydra by Embryo Microinjection

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among multicellular animals. Hydra is used as a model for the study of regeneration, pattern formation, and stem cells. However, research efforts have been hampered by lack of a reliable method for gene perturbations to study molecular function. The development of transgenic methods has revitalized the study of Hydra biology1. Transgenic Hydra allow for the tracking of live cells, sorting to yield pure cell populations for biochemical analysis, manipulation of gene function by knockdown and over-expression, and analysis of promoter function. Plasmid DNA injected into early stage embryos randomly integrates into the genome early in development. This results in hatchlings that express transgenes in patches of tissue in one or more of the three lineages (ectodermal epithelial, endodermal epithelial, or interstitial). The success rate of obtaining a hatchling with transgenic tissue is between 10% and 20%. Asexual propagation of the transgenic hatchling is used to establish a uniformly transgenic line in a particular lineage. Generating transgenic Hydra is surprisingly simple and robust, and here we describe a protocol that can be easily implemented at low cost.

Generation of Transgenic Hydra by Embryo Microinjection

Stably transgenic Hydra are made by microinjection of plasmid DNA into embryos followed by random genomic integration and asexual propagation to establish a uniform line. Transgenic Hydra are used to track cell movements, overexpress genes, study promoter function, or knock down gene expression using RNAi.

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is an alternative approach to chromatin immunoprecipitation (ChIP). An expressed fusion protein consisting of the protein of interest and the E. coli DNA adenine methyltransferase can methylate the adenine base in GATC motifs near the sites of protein-DNA interactions. Adenine-methylated DNA fragments can then be specifically amplified and detected. The original DamID assay detects the genomic locations of methylated DNA fragments by hybridization to DNA microarrays, which is limited by the availability of microarrays and the density of predetermined probes. In this paper, we report the detailed protocol of integrating high throughput DNA sequencing into DamID (DamID-seq). The large number of short reads generated from DamID-seq enables detecting and localizing protein-DNA interactions genome-wide with high precision and sensitivity. We have used the DamID-seq assay to study genome-nuclear lamina (NL) interactions in mammalian cells, and have noticed that DamID-seq provides a high resolution and a wide dynamic range in detecting genome-NL interactions. The DamID-seq approach enables probing NL associations within gene structures and allows comparing genome-NL interaction maps with other functional genomic data, such as ChIP-seq and RNA-seq.

We describe herein an assay by coupling DNA adenine methyltransferase identification (DamID) to high throughput sequencing (DamID-seq). This improved method provides a higher resolution and a wider dynamic range, and allows analyzing DamID-seq data in conjunction with other high throughput sequencing data such as ChIP-seq, RNA-seq, etc.

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is ...

Comparison of Three Different Methods for Determining Cell Proliferation in Breast Cancer Cell Lines

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility with high-throughput formatting. This protocol describes the use of three different methods for measuring cell proliferation in vitro including conventional hemocytometer counting chamber, a luminescence-based assay that utilizes the change in the metabolic activity of viable cells as a measure of the relative number of cells, and a multi-mode cell imager that measures cell number using a counting algorithm. Each method presents its own advantages and disadvantages for the measurement of cell proliferation, including time, cost and high-throughput compatibility. This protocol demonstrates that each method could accurately measure cell proliferation over time, and was sensitive to detect growth at differing cellular densities. Additionally, measurement of cell proliferation using a cell imager was able to provide further information such as morphology, confluence and allowed for a continual monitoring of cell proliferation over time. In conclusion, each method is capable of measuring cell proliferation, but the chosen method is user-dependent.

This protocol describes the use of three different methods for analyzing cell proliferation in breast cancer cell lines. This includes the use of conventional cell counting, luminescence-based cell viability, and cell counting through the use of a cell imager. Each offers advantages for the reproducible measurement of cell proliferation.

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility ...