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 N. benthamiana 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><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>&amp;beta;-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.

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...

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

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.

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 ...

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

Research

JoVE Journal

Biology

884 Views.  Shandong First Medical University. 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.

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

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Biochemistry

Expression and Purification of Mammalian Bestrophin Ion Channels

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- channel (CaCC) predominantly expressed in retinal pigment epithelium (RPE). The physiological and pathological significance of BEST1 is highlighted by the fact that over 200 distinct mutations in the BEST1 gene have been genetically linked to a spectrum of at least five retinal degenerative disorders, such as Best vitelliform macular dystrophy (Best disease). Therefore, understanding the biophysics of bestrophin channels at the single-molecule level holds tremendous significance. However, obtaining purified mammalian ion channels is often a challenging task. Here, we report a protocol for the expression of mammalian bestrophin proteins with the BacMam baculovirus gene transfer system and their purification by affinity and size-exclusion chromatography. The purified proteins have the potential to be utilized in subsequent functional and structural analyses, such as electrophysiological recording in lipid bilayers and crystallography. Importantly, this pipeline can be adapted to study the functions and structures of other ion channels.

The purification of ion channels is often challenging, but once achieved, it can potentially allow in vitro investigations of the functions and structures of the channels. Here, we describe the stepwise procedures for the expression and purification of mammalian bestrophin proteins, a family of Ca2+-activated Cl- channels.

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- ...

Preparation of Chloroplast Sub-compartments from Arabidopsis for the Analysis of Protein Localization by Immunoblotting or Proteomics

Chloroplasts are major components of plant cells. Such plastids fulfill many crucial functions, such as assimilation of carbon, sulfur and nitrogen as well as synthesis of essential metabolites. These organelles consist of the following three key sub-compartments. The envelope, characterized by two membranes, surrounds the organelle and controls the communication of the plastid with other cell compartments. The stroma is the soluble phase of the chloroplast and the main site where carbon dioxide is converted into carbohydrates. The thylakoid membrane is the internal membrane network consisting of grana (flat compressed sacs) and lamellae (less dense structures), where oxygenic photosynthesis takes place. The present protocol describes step by step procedures required for the purification, using differential centrifugations and Percoll gradients, of intact chloroplasts from Arabidopsis, and their fractionation, using sucrose gradients, in three sub-compartments (i.e., envelope, stroma, and thylakoids). This protocol also provides instructions on how to assess the purity of these fractions using markers associated to the various chloroplast sub-compartments. The method described here is valuable for subplastidial localization of proteins using immunoblotting, but also for subcellular and subplastidial proteomics and other studies.

Here, we describe a method to purify intact chloroplasts from Arabidopsis leaves and their three main sub-compartments (envelope, stroma, and thylakoids), using a combination of differential centrifugations, continuous Percoll gradients, and discontinuous sucrose gradients. The method is valuable for subplastidial and subcellular localization of proteins by immunoblotting and proteomics.

Chloroplasts are major components of plant cells. Such plastids fulfill many crucial functions, such as assimilation of carbon, sulfur and nitrogen as ...

Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall process costs. Alternative, cost-effective capture steps are therefore valuable for industrial-scale manufacturing, where large quantities of a single mAb are produced. Here we present a method for the immobilization of a DsRed-based epitope ligand to a cross-linked agarose resin allowing the selective capture of the HIV-neutralizing antibody 2F5 from crude plant extracts without using Protein A. The linear epitope ELDKWA was first genetically fused to the fluorescent protein DsRed and the fusion protein was expressed in transgenic tobacco (Nicotiana tabacum) plants before purification by immobilized metal-ion affinity chromatography. Furthermore, a method based on activated cross-linked agarose was optimized for high ligand density, efficient coupling and low costs. The pH and buffer composition and the soluble ligand concentration were the most important parameters during the coupling procedure, which was improved using a design-of-experiments approach. The resulting affinity resin was tested for its ability to selectively bind the target mAb in a crude plant extract and the elution buffer was optimized for high mAb recovery, product activity and affinity resin stability. The method can easily be adapted to other antibodies with linear epitopes. The new resins allow gentler elution conditions than Protein A and could also reduce the costs of an initial capture step for mAb production.

In this procedure, a DsRed-based epitope ligand is immobilized to produce a highly selective affinity resin for the capture of monoclonal antibodies from crude plant extracts or cell culture supernatants, as an alternative to Protein A.

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall ...

Recombinant Production of Bifidobacterial Endoglycosidases for N-glycan Release

Protein glycosylation is a diverse and common post-translational modification that has been associated with many important roles such as protein function, including protein folding, stability, enzymatic protection, and biological recognition. N-glycans attached to glycoproteins (such as lactoferrin, lactadherin, and immunoglobulins) cannot be digested by the host and reach the large intestine, where they are consumed by certain beneficial microbes. Therefore, they are considered next-generation prebiotic compounds that can selectively stimulate the gut microbiome's beneficial microorganisms. However, the isolation of these new classes of prebiotics requires novel enzymes. Here, we describe the recombinant production of novel glycosidases from different Bifidobacteria strains (isolated from infants, rabbits, chicken, and bumblebee) for improved N-glycan isolation from glycoproteins. The method presented in this study includes the following steps: molecular cloning of Bifidobacterial genes by an in vivo recombinational cloning strategy, control of transformation success, protein induction, and protein purification.

Recombinant Production of Bifidobacterial Endoglycosidases for N-glycan Release

Bifidobacteria possess a unique genomic capability for N-glycan cleavage. Recombinantly producing these enzymes would be a promising novel tool to release bioactive N-glycans from glycoprotein-rich substrates such as colostrum.

Protein glycosylation is a diverse and common post-translational modification that has been associated with many important roles such as protein function, ...

Assaying for Inorganic Polyphosphate in Bacteria

Inorganic polyphosphate (polyP) is a biological polymer found in cells from all domains of life, and is required for virulence and stress response in many bacteria. There are a variety of methods for quantifying polyP in biological materials, many of which are either labor-intensive or insensitive, limiting their usefulness. We present here a streamlined method for polyP quantification in bacteria, using a silica membrane column extraction optimized for rapid processing of multiple samples, digestion of polyP with the polyP-specific exopolyphosphatase ScPPX, and detection of the resulting free phosphate with a sensitive ascorbic acid-based colorimetric assay. This procedure is straightforward, inexpensive, and allows reliable polyP quantification in diverse bacterial species. We present representative polyP quantification from the Gram-negative bacterium (Escherichia coli), the Gram-positive lactic acid bacterium (Lactobacillus reuteri), and the mycobacterial species (Mycobacterium smegmatis). We also include a simple protocol for nickel affinity purification of mg quantities of ScPPX, which is not currently commercially available.

We describe a simple method for rapid quantification of inorganic polyphosphate in diverse bacteria, including Gram-negative, Gram-positive, and mycobacterial species.

Inorganic polyphosphate (polyP) is a biological polymer found in cells from all domains of life, and is required for virulence and stress response in many ...

Immunology and Infection

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 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.

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 ...

A Comparative Analysis of Recombinant Protein Expression in Different Biofactories: Bacteria, Insect Cells and Plant Systems

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the flexible, low-cost production of high-quality, bioactive products.
In this study, we compared the expression of a target human recombinant protein in traditional fermenter-based cell cultures (bacterial and insect) with plant-based expression systems, both transient and stable.
For each platform, we described the set-up, optimization and length of the production process, the final product quality and the yields and we evaluated provisional production costs, specific for the selected target recombinant protein.
Overall, our results indicate that bacteria are unsuitable for the production of the target protein due to its accumulation within insoluble inclusion bodies. On the other hand, plant-based systems are versatile platforms that allow the production of the selected protein at lower-costs than Baculovirus/insect cell system. In particular, stable transgenic lines displayed the highest-yield of the final product and transient expressing plants the fastest process development. However, not all recombinant proteins may benefit from plant-based systems but the best production platform should be determined empirically with a case-by-case approach, as described here.

In this study the expression of a target human recombinant protein in different production platforms was compared. We focused on traditional fermenter-based cultures and on plants, describing the set-up of each system and highlighting, on the basis of the reported results, the inherent limits and advantages for each platform.

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the ...

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 ...

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

Summary

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Chapters in this video

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Expression and Purification of Mammalian Bestrophin Ion Channels

Preparation of Chloroplast Sub-compartments from Arabidopsis for the Analysis of Protein Localization by Immunoblotting or Proteomics

Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study

Recombinant Production of Bifidobacterial Endoglycosidases for N-glycan Release

Assaying for Inorganic Polyphosphate in Bacteria

High-throughput Purification of Affinity-tagged Recombinant Proteins

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

A Comparative Analysis of Recombinant Protein Expression in Different Biofactories: Bacteria, Insect Cells and Plant Systems

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