This work is supported by the Youth Innovation Promotion Association CAS (2021084) and the Key Deployment Project Support Fund of the Three-Year Action Plan of the Institute of Microbiology, Chinese Academy of Sciences.

1. Preparation of N. benthamiana plants
<ol>
	<li>Place seedling blocks in a tray, add 1 L of water and soak for approximately 2 h until fully saturated.</li>
	<li>Evenly sprinkle wild-type N. benthamiana seeds on the seedling blocks, cover with a lid, and germinate for 1 week. Grow N. benthamiana in a greenhouse with 18 h photoperiod, 24 &#176;C, 45% relative humidity, and fertilize every 2 weeks.</li>
	<li>Once the seeds have sprouted, use tweezers to gently remove a seedling, taking care not to cause damage to its roots. Dig a small pit in the center of a new tray, place the seedling in it, and cover the roots lightly with soil. Cover with a lid and continue to grow.</li>
	<li>Grow till the seedlings have three leaves, with the lid on. Remove the lid when the 4th true leaf emerges.</li>
	<li>Allow plants to continue growing for two more weeks before agroinfiltration.</li>
</ol>
2. Construction of the recombinant GFP-His
<ol>
	<li>Perform N. benthamiana codon optimization and synthesis of GFP and PR1a (MGFVLFSQLPSFLLVSTLLLFLVISHSCRAG) based on the gene sequences.</li>
	<li>Carry out double digestion of pCAMBIA1300 vector using Xba I and Sac I enzymes (see Table of Materials).</li>
	<li>Perform amplification of PR1a using upstream primers (TTGGAGAGAACACGGGGGACTCTAGAATGGGATT 
	TGTTCTCTTTTC) and downstream primers (TCCTCG 
	CCCTTGCTCACCATGTCGACCCCGGCACGGCAAGA 
	GTGGG), amplification of GFP using upstream primers (CCCACTCTTGCCGTGCCGGGGTCGACATGGTGAG 
	CAAGGGCGAGGA) and downstream primers (GGGGAAATTCGAGCTCTCAGTGGTGATGGTGGTG 
	GTGAGATCTTCCGGACTTGT).</li>
	<li>Using homologous recombination, clone PR1a, and GFP into the pCAMBIA1300 plant expression vector. After transferring into E. coli, select single clones and send them to the company for sequencing. Comparison of sequencing results and the right plasmid is the recombinant plasmid pCAMBIA1300-PR1a-GFP-His, referred to as p35s-PR1a-GFP-His.</li>
</ol>
3. Production of GFP-His in N. benthamiana
<ol>
	<li>Add 1 &#181;L of GFP plasmid into 50 &#181;L of Agrobacterium receptor cells GV3101, mix gently and add to the electrode cup, remove after electrocution on the electroshocker.</li>
	<li>Spread transformed Agrobacterium tumefaciens on an LB solid medium plate containing 50 &#181;g/mL kanamycin (Kan) and 50 &#181;g/mL rifampicin (Rif). Incubate inverted at 28 &#176;C for 48 h. The pCAMBIA1300 vector is kan-resistant, and Agrobacterium is rif-resistant; only the correct Agrobacterium can grow on plates with kan and rif. The monoclonal clones were the positive transformants, named A. tumefaciens GV3101-p35s-PR1a-GFP-His (AtGPG).</li>
	<li>Use a sterilized toothpick to pick up an appropriate amount of AtGPG and inoculate it into 4 mL of liquid LB with Kan and Rif. Culture at 28 &#176;C, 220-300 rpm for 24 h.</li>
	<li>Subculture bacteria 1:50 into fresh LB (50 &#181;g/mL Kan, 10 mM 2-(N-Morpholino) ethanesulfonic acid (MES), 20 &#181;M acetosyringone (AS)) and incubate at 28 &#176;C, 220-300 rpm for 10-12 h.</li>
	<li>Harvest bacteria by centrifugation at 1500 x g for 10 min and resuspend the pellet with 1 mL of MMA (10 mM MgCl2, 10 mM MES pH 5.6, 100 &#181;M AS).</li>
	<li>Measure and adjust the optical density (OD) of AtGPG to OD600=0.8 using MMA. Incubate the bacteria for another 2-4 h at room temperature. Draw up the bacterial solution with a needleless syringe, press it against the back of the leaves lightly, and give pressure to penetrate the solution into the leaves. Inject only the middle 3-4 leaves per plant.</li>
	<li>Continue to grow N. benthamiana in the greenhouse for 3-4 days before harvesting leaves for protein extraction.</li>
</ol>
4. Observing subcellular localization of GFP
<ol>
	<li>After 48 h of agroinfiltration, select a flat leaf and use a 6 mm hole punch to clamp the blade for punching.</li>
	<li>Drip 30% sucrose solution onto a glass slide, place the leaf sample with the back facing upwards and cover it with a cover glass.</li>
	<li>Set the excitation wavelength to 488 nm and detect the green fluorescence at 490-550 nm after magnification of 400x. Take the first photo once the slide is prepared and another one 10 min later.</li>
</ol>
5. Extracting GFP from plant
<ol>
	<li>AWF extraction
	<ol>
		<li>Collect the intact fresh N. benthamiana leaves expressing recombinant GFP at 3 days post agroinfiltration. Treat the leaf gently and maintain leaf integrity to avoid intracellular protein efflux. Use collected leaves for the next step immediately to avoid protein degradation.</li>
		<li>Rinse leaf blade with pre-cooled sterilized ddH2O to remove debris.</li>
		<li>Weigh fresh leaves and submerge 20 g leaves (abaxial side down) in 100 mL of pre-cooled 100 mM phosphate buffer (PB 100 mM Na2HPO4 and 100 mM NaH2PO4 were separately configured and mixed at a ratio of 2.2:1 pH 7.0) in a 200 mL wide-mouth beaker. Cover leaves with 100 mesh nylon fine mesh yarn (see Table of Materials) and a plastic plate. 
		NOTE: Pre-cooling prevents protein degradation. Buffer volumes can be adjusted based on the experimental scale. Ensure all leaves are immersed.</li>
		<li>Place the beaker into a vacuum pump. Apply 0.8 MPa vacuum for 1 min, then quickly restore to atmospheric pressure.</li>
		<li>Remove leaves from the buffer and gently blot dry with absorbent paper. Roll up leaves, wrap with mesh yarn, and place 5 g of leaves into each 50 mL centrifuge tube. To collect AWF, position rolled leaves tip-side up in tubes with mesh yarn fastened by the tube caps.</li>
		<li>Centrifuge at 4 &#176;C, 500 x g for 5 min. Remove leaves and collect the AWF at the bottom of the tube using a pipette. About 5 mL of AWF can be obtained from each tube. 
		NOTE: In order to minimize interference from intracellular proteins and to extract the maximum amount of AWF proteins, the extraction can be repeated 2x-3x, but not more than 3x.</li>
	</ol>
	</li>
	<li>Total soluble protein extraction
	<ol>
		<li>Grind 20 g leaves into fine powder in liquid nitrogen. Add extraction buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 0.034% &#946;-mercaptoethanol (see Table of Materials)) at a 1: 2 ratio.</li>
		<li>Shake homogenate in a vertical mixer for 30 min at 4 &#176;C. Centrifuge homogenate at 13,000 x g for 15 min at 4 &#176;C.</li>
		<li>Transfer the supernatant to a new tube and repeat centrifugation for 15 min. Transfer the cleared supernatant to another tube.</li>
		<li>Filter the supernatant through a 0.22 &#181;m filter (see Table of Materials) to obtain total soluble protein extract.</li>
	</ol>
	</li>
</ol>
&#160;&#160;
6. Purification of GFP-His with nickel (Ni) affinity chromatography
NOTE: Perform all steps at 4 &#176;C.
<ol>
	<li>Add Ni sepharose excel resin (see Table of Materials) to a tube in a 1:1000 ratio relative to the protein extract volume from step 5.1.6. Resin volume equals 1/1000th of the protein extract volume.</li>
	<li>Wash Ni resin 3x with 10x resin volume of ddH2O and PB (pH 7.0) separately in turn and remove the supernatant.</li>
	<li>Incubate protein extract with Ni resin for 2 h to allow full binding. Centrifuge at 500 x g for 5 min to remove unbound proteins in the supernatant. Wash 3x with 20x resin volume of PB. Centrifuge at 500 x g for 5 min and remove the supernatant.</li>
	<li>Elute GFP-His by adding 10x resin volume of 250 mM imidazole (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 10% glycerol, 250 mM imidazole) and collect the supernatant.</li>
</ol>
7. Coomassie blue staining of proteins
<ol>
	<li>Add 10 &#181;L of 5x SDS loading buffer to 40 &#181;L of protein samples, boil for 10 min, then run on 15% SDS-PAGE at 110 V for 60 min.</li>
	<li>Stain protein gels with Coomassie blue solution (0.1% R-250, 25% isopropanol, 10% Glacial acetic acid) for 2 h.</li>
	<li>Pour off the solution and wash with decolorizing solution for 2 h. Observe the gel; proteins on the gel will be colored blue.</li>
</ol>
8. Western blotting of proteins
<ol>
	<li>Add 10 &#181;L of 5x SDS loading buffer to 40 &#181;L of protein samples, boil for 10 min, then run on 15% SDS-PAGE at 110 V for 60 min.</li>
	<li>Transfer proteins to a 0.45 &#181;m nitrocellulose membrane at 280 mA. Block the membrane with 5% skimmed milk for 1 h.</li>
	<li>Dilute the anti-His-tag antibodies at 1:5000 and incubate the membrane with the antibodies for 1 h. Use the tris-buffered saline and tween 20 (TBST) to wash the membrane 4x for 5 min.</li>
	<li>Dilute the anti-mouse antibodies at 1:10000 and incubate the membrane with the antibodies for 1 h. Use TBST to wash the membrane 4x for 5 min.</li>
	<li>Mix the developer (see Table of Materials) in the ratio of 1:1. Add 400 &#181;L to the membrane and allow to react for 1 min. Take photographs after 1 min exposure with a visualizer.</li>
</ol>
9. Protein concentration assay
<ol>
	<li>Add 5 &#181;L of protein to 1 mL of Bradford 1x dye reagent and react for 5 min.</li>
	<li>Determine the OD595 value after zeroing and calculate the protein concentration from the standard curve.</li>
</ol>
10. Protein quantification&#160;31
<ol>
	<li>Perform Western blotting of protein samples together with standards.</li>
	<li>Perform quantification of GFP by ratio to standards using ImageJ 1.5.0 greyscale analysis.</li>
</ol>
&#160;&#160;

Efficient Extraction and Purification of GFP-His from &lt;i&gt;N. benthamiana&lt;/i&gt; Leaves

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The authors declare no conflict of interest.

Using plants to produce therapeutic proteins has expanded quickly in recent years1,2,3,4,5,6. Protein-encoding genes are cloned into expression vectors and delivered into plant tissues via agroinfiltration. After production by plant cells, proteins are purified for downstream applications. Typically, recombinant proteins are ...

Nowadays, various plant-produced recombinant therapeutic proteins are under study, including antibodies, vaccines, bioactive proteins, enzymes, and small polypeptides1,2,3,4,5,6. Plants are becoming an increasingly utilized platform for producing therapeutic proteins due to their safety, low cost, and ability to rapidly scale production7,8. Protein expression and modification in plant systems, along with protein purification, are critical factors determining the productivity of plant bioreactors9,10. Enhancing plant productivity and obtaining high-purity target proteins are core challenges facing plant-based production systems11,12.
The subcellular localization and modification of recombinant proteins depends on the specific signal peptide encoded at the N-terminus, which targets the protein to its final organelle destination during gene expression. Recombinant proteins are designed to localize to the apoplast, endoplasmic reticulum (ER), chloroplast, vacuole, or cytoplasm, based on their unique properties13. For antigens and antibodies, secreted proteins are preferred. Before secretion, proteins progress through ER and Golgi for correct folding and post-translational modifications14,15,16.
After production in plants, recombinant proteins must be purified from plant extracts. Typically, proteins are purified from total soluble plant extracts, which contain abundant intracellular proteins, misfolded and unmodified products, and cytochromes17. To remove impurities, plant extracts undergo extensive fractionation, including ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO)-specific affinity chromatography, phytate precipitation, polyethylene glycol precipitation, and adjustment of temperature and pH18,19. Such impurity-removal steps are laborious and can compromise protein quality.
The plant cell compartment beyond the plasma membrane is defined as the apoplast, encompassing the cell wall, intercellular spaces, and xylem20. The aqueous phase is commonly called apoplast washing fluid (AWF). AWF contains molecules secreted by cells to regulate numerous biological progresses like transport, cell wall metabolism, and stress responses21,22. In contrast to TSP, the molecular constituents of AWF are less complex. Extracting recombinant proteins from AWF may avoid contamination by intracellular proteins, misfolded products, and cytoplasmic remnants. Briefly, extraction lipid enters leaves through the stomata under negative pressure, and the AWF is collected by gentle centrifugation23. While isolation of AWF is widely employed to study the biological progress inside apoplast24,25,26,27, it has not been exploited in plant-based production systems.
Improving plant productivity and obtaining high purity target proteins are central challenges for plant production systems28. Typically, proteins are purified from total soluble plant extracts containing large amounts of intracellular proteins, misfolded and unmodified products, and cytochromes29, which require great costs for subsequent purification30. The use of AWF protein extraction methods for the extraction of exogenous recombinant proteins secreted into apoplast can effectively improve the homogeneity of recombinant proteins and reduce the cost of protein purification. Here, we use a signal peptide PR1a to enable exogenous protein secretion into the apoplast space and introduce a detailed method for recombinant protein purification from the AWF of N. benthamiana.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 mesh nylon fine mesh yarn</td>
			<td>Guangzhou Huayu Trading limited company</td>
			<td>hy-230724-2</td>
			<td>For AWF protein extraction</td>
		</tr>
		<tr>
			<td>50 ml centrifuge tubes</td>
			<td>Vazyme</td>
			<td>TCF00150</td>
			<td>For centrifuge</td>
		</tr>
		<tr>
			<td>ClonExpress II one step cloning kit</td>
			<td>Vazyme</td>
			<td>C112-02</td>
			<td>For clone</td>
		</tr>
		<tr>
			<td>FastDigest Sac1</td>
			<td>NEB</td>
			<td>R3156S</td>
			<td>For clone</td>
		</tr>
		<tr>
			<td>FastDigest XbaI</td>
			<td>NEB</td>
			<td>FD0685</td>
			<td>For clone</td>
		</tr>
		<tr>
			<td>ImageJ 1.5.0</td>
			<td>/</td>
			<td>/</td>
			<td>For greyscale analysis</td>
		</tr>
		<tr>
			<td>Large filter</td>
			<td>Thermo Fisher</td>
			<td>NEST</td>
			<td>For filtering</td>
		</tr>
		<tr>
			<td>Laser scanning confocal microscopy</td>
			<td>Leica SP8</td>
			<td></td>
			<td>For subcellular localization</td>
		</tr>
		<tr>
			<td>Ni sepharose excel</td>
			<td>Cytiva</td>
			<td>10339806</td>
			<td>For protein purification</td>
		</tr>
		<tr>
			<td>Precast protein plus gel</td>
			<td>YEASEN</td>
			<td>36246ES10</td>
			<td>For SDS-PAGE</td>
		</tr>
		<tr>
			<td>Small filter</td>
			<td>Nalgene</td>
			<td>1660045</td>
			<td>For filtering</td>
		</tr>
		<tr>
			<td>SuperSignal West Femto Substrate Trial Kit</td>
			<td>Thermo Fisher</td>
			<td>34076</td>
			<td>For western blotting</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>SCR</td>
			<td>30188928</td>
			<td>To configure the extraction buffer</td>
		</tr>
		<tr>
			<td>Type rotaryvang vacuum pump</td>
			<td>Zhejiang taizhouqiujing vacuum pump limited company</td>
			<td>2XZ-2</td>
			<td>For vacuum infiltration</td>
		</tr>
		<tr>
			<td>Vertical mixer</td>
			<td>Haimen Qilinbel Instrument Manufacturing limited company</td>
			<td>BE-1200</td>
			<td>For mixing</td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>SIGMA</td>
			<td>BCBK8223V</td>
			<td>To configure the extraction buffer</td>
		</tr>
	</tbody>
</table>

apoplast-extraction based method to improve the purity of plant produced recombinant protein  

Plants are a newly developing eukaryotic expression system being explored to produce therapeutic proteins. Purification of recombinant proteins from plants is one of the most critical steps in the production process. Typically, proteins were purified from total soluble proteins (TSP), and the presence of miscellaneous intracellular proteins and cytochromes poses challenges for subsequent protein purification steps. Moreover, most therapeutic proteins like antigens and antibodies are secreted to obtain proper glycosylation, and the presence of incompletely modified proteins leads to inconsistent antigen or antibody structures. This work introduces a more effective method to obtain highly purified recombinant proteins from the plant apoplastic space. The recombinant Green fluorescent protein (GFP) is engineered to be secreted into the apoplast of Nicotiana benthamiana and is then extracted using an infiltration-centrifugation method. The GFP-His from the extracted apoplast is then purified by nickel affinity chromatography. In contrast to the traditional methods from TSP, purification from the apoplast produces highly purified recombinant proteins. This represents an important technological improvement for plant production systems.

Author Spotlight: Optimizing Apoplast Protein Extraction for Efficient Recovery of Recombinant Proteins from Plant Cells

Apoplast-Extraction Based Method to Improve the Purity of Plant Produced Recombinant Protein  

We investigated how to extract recombinant proteins from plant cells more efficiently. We answered the question of whether the apoplast protein extraction method is feasible when applied to the recombinant proteins. This extraction method extracts proteins secreted into the apoplast, so they have homogeneity.There is no need for cell disruption, which reduce the interference from intracellular proteins and the subsequent purification costs. We will focus on how to increase the secretion of recombinant proteins in the apoplast space to further improve the efficiency of the apoplast protein extraction method.

GFP-His is targeted for secretion into the apoplastic space of N. benthamiana 
GFP was cloned into the pCAMBIA1300 plasmid with an N-terminal PR1a signal peptide for secretion and a C-terminal His-tag for purification, generating p35s-PR1a-GFP-His (Figure 1A). The recombinant protein was transiently expressed in N. benthamiana and a 30 kDa band was detected by western blot using anti-His antibody at 3 days post agroinfiltration <strong...

The purification of recombinant proteins from plant systems is usually challenged by plant proteins. This work provides a method to effectively extract and purify a secreted recombinant protein from the apoplast of Nicotiana benthamiana.

Plants are a newly developing eukaryotic expression system being explored to produce therapeutic proteins. Purification of recombinant proteins from ...

apoplast-extraction-based-method-to-improve-purity-plant-produced

Research

JoVE Journal

Biology

666 Views.  Shandong First Medical University. We investigated how to extract recombinant proteins from plant cells more efficiently. We answered the question of whether the apoplast protein extraction method is feasible when applied to the recombinant proteins. This extraction method extracts proteins secreted into the apoplast, so they have homogeneity.There is no need for cell disruption, which reduce the interference from intracellular proteins and the subsequent purification costs. We will focus on how to increase the secretio...

Video: Author Spotlight: Optimizing Apoplast Protein Extraction for Efficient Recovery of Recombinant Proteins from Plant Cells

Extraction and Purification of a Secreted Recombinant Protein from the Apoplast of Nicotiana benthamiana

To observe subcellular localization of GFP, after 48 hours of agroinfiltration of Nicotiana benthamiana, select a flat leaf and make a six-millimeter hole using a hole punch. Drip 30%sucrose solution onto a glass slide. Place the leaf sample with the back facing upwards, and cover it with a cover glass.Collect the intact, fresh N.benthamiana leaves expressing recombinant GFP at three days post-agroinfiltration, and weigh the leaves. Rinse the leaf blade with pre-cooled, sterilized, double-distilled water to remove debris. Submerge 20 grams with the abaxial side down in 500 milliliters of pre-cooled 100-millimolar phosphate buffer in a 1, 000 milliliter wide-mouth beaker.Cover the leaves with 100 mesh nylon, fine mesh yarn and a plastic plate. Connect the beaker to a vacuum pump. Apply 0.8 megapascal vacuum for one minute, then quickly restore to atmospheric pressure.Remove leaves from the buffer and gently blot dry with absorbent paper. Roll up the leaves and wrap them with mesh yarn. For collecting apoplastic washing fluid, or AWF, position rolled leaves tip-side up in 50-milliliter centrifuge tubes with mesh yarn fastened by the tube caps.Centrifuge the leaves at 500g for five minutes at four degrees Celsius. Remove the leaves and collect AWF using a pipette. Add Nickel Sepharose excel resin to a tube in a one-to-1, 000 ratio relative to the protein extract volume.Wash nickel resin three times with 10 times the resin volume of double-distilled water and phosphate buffer separately, in turn. Incubate protein extract with nickel resin for two hours to allow full binding. Then, centrifuge at 500g for five minutes at four degrees Celsius to remove unbound proteins in the supernatant.Wash three times with 20 times the resin volume of phosphate buffer. After the last wash, add 10 times the resin volume of 250 millimolar imidazole to elute GFPs. Centrifuge the mixture at 500g for five minutes at four degrees Celsius, and collect the supernatant.Western blot analysis confirmed the transient expression of 30 kilodalton recombinant protein GFPs in N.benthamiana. Fluorescence microscopy illustrated the presence of GFPs within the apoplast. AWF proteins extracted by vacuum infiltration centrifugation also contained GFPs.Out of the eight extraction solutions tested, phosphate buffer extracted the greatest amount of AWF and GFPs. Comparing phosphate buffer at varying pH revealed improved AWF and GFPs extraction at neutral to mildly alkaline conditions. Additionally, 100 millimolar phosphate buffer showed maximal efficacy for recovering AWF and GFPs.With a 100-millimolar phosphate buffer, 495 micrograms of total AWF proteins per gram of fresh leaf were recovered from the apoplast. The extracted AWF contained 10 micrograms of GFPs, which corresponded to approximately 18%of the GFPs in the total soluble protein. Following nickel affinity chromatography, GFPs'purity reached 84.3%from AWF extraction, substantially higher than the 44.9%purity achieved through total soluble protein extraction.