Name: biosynthesis of a flavonol from a flavanone by establishing a one-pot bienzymatic cascade
Uploaded: 2019-08-14T12:30:00
Description: Watch this Scientific Journal Video about Biosynthesis of a Flavonol from a Flavanone by Establishing a One-pot Bienzymatic Cascade at JoVE.com

This work was financially supported by Yangzhou University Specially-Appointed Professor Start-up Funds, Jiangsu Specially-Appointed Professor Start-up Funds, Six Talent Peaks Project in Jiangsu Province (Grant No. 2014-SWYY-016), and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (Veterinary Medicine). We thank the Testing Center of Yangzhou University for HPLC and MS analyses of flavonoids.

1. Isolate total RNA from plant tissues26,27
<ol>
	<li>Homogenize the plant tissues.
	<ol>
		<li>Collect 100 mg of a fresh plant tissue (e.g., 4-week-old seedlings from Arabidopsis thaliana). Freeze the tissue and a pestle and mortar with liquid nitrogen, followed by grinding the tissue into powder.</li>
		<li>Add 1 mL of RNA isolation reagent (see Table of Materials) into the mortar. The reagent will be frozen immediately. Homogenize ...

<ol>
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Quite a number of studies are focused on the derivation of flavonols due to their potential application in health care and food industry. However, traditional plant extraction using organic solvents and chemical synthesis possess intrinsic disadvantages, which restrict their use in the production of flavonols. Here, we report a detailed method for producing a flavonol from a flavanone in one pot by establishing an in vitro bienzymatic cascade. The critical steps in this protocol are: 1) obtaining pure recombinant enzymes...

Flavonols are a major subclass of plant flavonoids and are involved in plant development and pigmentation1,2,3. More importantly, these compounds possess a wide range of health-beneficial activities, such as anti-cancer4,5, anti-oxidative6, anti-inflammatory7, antiobesity8, anti-hypertensive9, and memory recall properties10, leading to a large number of studies on these plant-derived secondary metabolites. Traditionally, these compounds are mainly derived from plant extraction using organic solvents. However, due to their very low contents in plants11,12,13, the production cost for most flavonols remains high, which imposes great restrictions on their application in healthcare and the food industry.
During the past decades, scientists have developed quite a number of methods to derive flavonoids14,15. However, chemical synthesis of these complicated molecules possesses a variety of intrinsic disadvantages16. It requires not only toxic reagents and extreme reaction conditions, but also many steps to produce a target flavonoid compound14,17. Moreover, another important challenge in this strategy is the chiral synthesis of active flavonoid molecules. Therefore, it is not an ideal strategy to produce flavonoids at a commercial scale via chemical synthesis16,17.
Recently, scientists have developed a promising alternative strategy to produce these complicated natural compounds by engineering microbes with a pathway for flavonoid biosynthesis18,19,20,21,22, which has been successfully deciphered in plants23. For example, Duan et al. introduced a biosynthetic pathway into the budding yeast Saccharomyces cerevisiae to produce kaempferol (KMF)24. Malla et al. produced astragalin, a glycosylated flavonol, by introducingflavanone 3-hydroxylase (f3h), flavonol synthase (fls1), and UDP-glucose:flavonoid 3-O-glucosyltransferase UGT78K1 genes into Escherichia coliBL21(DE3)17. Even though there are quite a few paradigms, not all genetically engineered microbes produce the products of interest due to the complexity of a cellular platform, the incompatibility between artificially synthesized genetic elements and hosts, the inhibitory effect of target products against host cells, and the instability of an engineered cellular system itself16.
Another promising alternative strategy for flavonoid production is to establish a multienzymatic cascade in vitro. Cheng et al. have reported that enterocin polyketides can be successfully synthesized by assembling a complete enzymatic pathway in one pot25. This cell-free synthetic strategy circumvents the restrictions of a microbial production factory and thus is feasible for producing some flavonoids in large quantity16.
Recently, we have successfully developed a bienzyme synthetic system to convert naringenin (NRN) into KMF in one pot16. Here, we describe this system in great details and the methods involved in analyzing the products. We also present two examples that use this system to produce KMF from NRN and quercetin (QRC) from eriodictyol (ERD). In addition, we discuss crucial steps of this method and future research directions in the biosynthesis of flavonoids.

<table>
	<tbody>
		<tr>
			<td>2&#215; Pfu MasterMix</td>
			<td>Beijing CoWin Biotech Co., Ltd</td>
			<td>CW0717A</td>
			<td>PCR amplification of genes with high fidelity</td>
		</tr>
		<tr>
			<td>Agilent 1200 Series RRLC system with an Agilent 6460 Triple Quadrupole LC/MS system</td>
			<td>Agilent Technologies, Inc</td>
			<td>N/A</td>
			<td>an equipment for analysis of flavonoids by HPLC/MS</td>
		</tr>
		<tr>
			<td>Agilent MassHunter Workstation (version B.03.01)</td>
			<td>Agilent Technologies, Inc</td>
			<td>N/A</td>
			<td>a software for collection of the data from the Agilent 1200 Series RRLC system with an Agilent 6460 Triple Quadrupole LC/MS system</td>
		</tr>
		<tr>
			<td>dihydrokaempferol</td>
			<td>Sigma-Aldrich Co. LLC</td>
			<td>91216</td>
			<td>intermediate product for producing kaempferol from naringenin</td>
		</tr>
		<tr>
			<td>dihydroquercetin</td>
			<td>Sichuan Provincial Standard Substance Center for Chinese Herbal Medicine</td>
			<td>PCS0371</td>
			<td>intermediate product for producing quercetin from eriodictyol</td>
		</tr>
		<tr>
			<td>DNA Clean-up Kit</td>
			<td>Beijing CoWin Biotech Co., Ltd</td>
			<td>CW2301</td>
			<td>purification of PCR-amplified or gel-purified DNA</td>
		</tr>
		<tr>
			<td>eriodictyol</td>
			<td>Shanghai Yuan Ye Biotechnology Co., Ltd.</td>
			<td>B21160</td>
			<td>substrate for producing quercetin</td>
		</tr>
		<tr>
			<td>Escherichia coli BL21(DE3)</td>
			<td>Beijing CoWin Biotech Co., Ltd</td>
			<td>CW0809</td>
			<td>bacteria strain for expressing target genes</td>
		</tr>
		<tr>
			<td>Escherichia coli DH5&#945;</td>
			<td>Beijing CoWin Biotech Co., Ltd</td>
			<td>CW0808</td>
			<td>bacteria strain for plasmid proliferation</td>
		</tr>
		<tr>
			<td>FreeZone 1 Liter Benchtop Freeze-Dry System</td>
			<td>Labconco Corporation</td>
			<td>7740020</td>
			<td>an equipment for freeze-drying of flavonoids dissolved in organic solvent</td>
		</tr>
		<tr>
			<td>Gel Extraction Kit</td>
			<td>Beijing CoWin Biotech Co., Ltd</td>
			<td>CW2302</td>
			<td>purification of a DNA band from an agarose gel</td>
		</tr>
		<tr>
			<td>Gel Imaging System</td>
			<td>Shanghai Tanon Science &#38; Technology Co. Ltd.</td>
			<td>Tanon- 
			2500</td>
			<td>an equipment for visualization of DNA band on an agarose gel or flavonoid spot on a polyamide TLC plate</td>
		</tr>
		<tr>
			<td>GenElute Plasmid Miniprep Kit</td>
			<td>Sigma-Aldrich Co. LLC</td>
			<td>PLN350-1KT</td>
			<td>minipreparation of plasmids</td>
		</tr>
		<tr>
			<td>kaempferol</td>
			<td>Sigma-Aldrich Co. LLC</td>
			<td>60010</td>
			<td>final reaction product and standard substance</td>
		</tr>
		<tr>
			<td>MassHunter Quanlitative Analysis (version B.01.04)</td>
			<td>Agilent Technologies, Inc</td>
			<td>N/A</td>
			<td>a software for analysis of HPLC/LC/MS data</td>
		</tr>
		<tr>
			<td>NanoDrop Microvolume UV-Vis Spectrophotometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>ND-8000-GL</td>
			<td>an equipment for determination of DNA/RNA concentration</td>
		</tr>
		<tr>
			<td>naringenin</td>
			<td>Sigma-Aldrich Co. LLC</td>
			<td>N5893</td>
			<td>substrate for producing kaempferol</td>
		</tr>
		<tr>
			<td>Ni-IDA Agarose Resin</td>
			<td>Beijing CoWin Biotech Co., Ltd</td>
			<td>CW0010</td>
			<td>purification of His-tagged fusion proteins</td>
		</tr>
		<tr>
			<td>pET-32a(+)</td>
			<td>Novagen</td>
			<td>69015-3</td>
			<td>plasmid for cloning and expressing target genes</td>
		</tr>
		<tr>
			<td>plasmid sequencing</td>
			<td>GENEWIZ Suzhou</td>
			<td>N/A</td>
			<td>sequencing of recombinant plasmids</td>
		</tr>
		<tr>
			<td>primer synthesis</td>
			<td>GENEWIZ Suzhou</td>
			<td>N/A</td>
			<td>synthesis of PCR primers</td>
		</tr>
		<tr>
			<td>quercetin</td>
			<td>Shanghai Aladdin Biochemical Technology Co.,Ltd.</td>
			<td>Q111273</td>
			<td>final reaction product and standard substance</td>
		</tr>
		<tr>
			<td>SuperRT cDNA Synthesis Kit</td>
			<td>Beijing CoWin Biotech Co., Ltd</td>
			<td>CW0741</td>
			<td>synthesis of the first strand of cDNA from total RNA</td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>Thermo Fisher Scientific</td>
			<td>EL0016</td>
			<td>ligation of an insert into a linearized vector DNA</td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>Thermo Fisher Scientific</td>
			<td>15596018</td>
			<td>isolation of total RNA</td>
		</tr>
		<tr>
			<td>Vector NTI Advance</td>
			<td>Thermo Fisher Scientific</td>
			<td>12605099</td>
			<td>a software for PCR primer design and DNA sequence analysis</td>
		</tr>
		<tr>
			<td>Xcalibur v2.0.7</td>
			<td>Thermo Fisher Scientific</td>
			<td>N/A</td>
			<td>a software for analysis of HPLC data</td>
		</tr>
	</tbody>
</table>

biosynthesis of a flavonol from a flavanone by establishing a one-pot bienzymatic cascade

Flavonols are a major subclass of flavonoids with a variety of biological and pharmacological activities. Here, we provide a method for the in vitro enzymatic synthesis of a flavonol. In this method, Atf3h and Atfls1, two key genes in the biosynthetic pathway of the flavonols, are cloned and overexpressed in Escherichia coli. The recombinant enzymes are purified via an affinity column and then a bienzymatic cascade is established in a specific synthetic buffer. Two flavonols are synthesized in this system as examples and determined by TLC and HPLC/LC/MS analyses. The method displays obvious advantages in the derivation of flavonols over other approaches. It is time- and labor-saving and highly cost-effective. The reaction is easy to be accurately controlled and thus scaled up for mass production. The target product can be purified easily due to the simple components in the system. However, this system is usually restricted to the production of a flavonol from a flavanone.

F3H and FLS1 are two important key enzymes in the conversion of a flavanone into a flavonol in plants as shown in Figure 1. To develop an in vitro biosynthetic system for producing a flavonol from a flavanone, Atf3h (GenBank accession no.NM_114983.3) and Atfls1 (GenBank accession no. NM_120951.3) genes were cloned from the seedlings of 4-week-old A. thaliana into a prokaryotic expression vector pET-32a(+). The recombinant plasmids w...

Watch this Scientific Journal Video about Biosynthesis of a Flavonol from a Flavanone by Establishing a One-pot Bienzymatic Cascade at JoVE.com

Biosynthesis of a Flavonol from a Flavanone by Establishing a One-pot Bienzymatic Cascade

The derivation of a flavonol is crucial for its application in healthcare and the food industry. Here, we provide a detailed protocol for the biosynthesis of a flavonol from a flavanone and discuss the crucial steps and its advantages over other approaches.

Flavonols are a major subclass of flavonoids with a variety of biological and pharmacological activities. Here, we provide a method for the in vitro ...

Using this protocol, we can easily produce quite a number of flavonols from flavanones in one pot. This technique is labor-and time-saving, highly cost-effective, and easy to control, so it has a huge industrialization potential. Yes, this method can provide insight into economic production of other secondary metabolites, but it needs some modifications when applied to it.He will usually get nothing or very low yield. My advice is to handle recombinant enzymes on ice and add 10%glycerin to the stock solution of enzymes. That's because only through visual demonstration can a new hand understand the important details and truth affecting the successful execution of the experiment.First prepare buffers. Make two times synthetic buffer by dissolving appropriate amount of TRIS-base, sodium ascorbate, alpha ketoglutaric acid into de-ionized water, and pipette appropriate amount of glycerol to the solution. Use a pipette to add hydrochloric acid to adjust the pH to 7.2, and adjust the volume by adding de-ionized water.Store the two times synthetic buffer at four degrees Celsius for future use. Then make a 100 times stock solution by dissolving ferrous sulfate heptahydrate in deionized water. Make a stock solution of 25 millimolar flavonoid by dissolving flavonoid in methanol, and store the solution at minus 20 degrees Celsius.The most critical step is to set up a synthetic system. To set up a synthetic system to produce a flavanol from a flavanone, first prepare the synthetic system in a two milliliter tube according to the reagents listed in this table. Place the open tube in a shaking heat block at 40 degrees Celsius, at 600 rpm for forty minutes.After that, terminate the reaction by adding 10 microliters of acetic acid, and 100 microliters of ethyl acetate. Two hours later, use a pipette to tranfer the top organic phase to a 1.5 milliliter tube, and place the tube in a hood for air drying at room temperature. To perform TLC analysis, first retrieve the tubes from the hood, and redissolve the flavanoid powder in 160 microliters of methanol in a tube.Prepare authentic flavonoid samples with serial concentrations, by diluting the 25 millimolar flavonoid stock solution in methanol. Load one microliter of the redissolved reaction sample, and one microliter of the authentic flavonoid sample, onto a polyamide plate. Do the same for the other concentrations of authentic flavonoid on the same plate.Then in a fume hood, put the sample loaded plate in a chromatography cylinder filled with a solvent system. Attach the plate to the bottom of the cylinder, and run the plate in the solvent system for 20 minutes. Air dry the plates at room temperature.With a sprayer bottle, spray the plates with 1%ethanolic solution of aluminum chloride, followed by air drying again at room temperature for 30 minutes. Visualize the spots on the plates under a UV light at 254 nanometers, and take images, then on the computer, open the software ImageJ. Click file, open, to open the image to be analyzed.Click the left most rectangular selection tool in the ImageJ user interface. Outline the ROI in the image with the mouse, and press one to label the first ROI. Move the rectangular selection with the mouse right to the next ROI, and press two to label the second ROI.Repeat to label all other ROIs by pressing two. Press three to generate profile plots for all ROIs, in a pop-up window. Then, use the straight line selection tool to drop baselines so as to define a closed area for each peak of interest.Activate the wand tool, by clicking the corresponding icon in the ImageJ user interface. Click inside the peak to display results for all peaks in a pop-up window. Next, in Excel, plot the gray values from the pop-up window against the corresponding flavonoid concentrations, to make a TLC based standard curve of the authentic flavonoid.Then calculate the yield of the flavonoid of interest produced in this protocol, according to the resulting formula. Use a syringe to process each sample sequentially through a 0.45 micrometer and 0.22 micrometer filters. Load the samples into an HPLC-MS system through suction nozzle, and separate the samples at 30 degrees Celsius using a C18 column.Elute the column at one milliliter per minute by a gradient of 10 to 85 volume per cent acetyl nitrile in water, and monitor the absorbance of the eluate from 200 to 800 nanometers. Perform the LC-MS analysis in a negative ion mode, with a drying nitrogen flow of 10 liters per minute at 300 degrees Celsius, in a sheath gas flow of seven liters per minute at 250 degrees Celsius, and collect data using a built in software. To extract single wavelength chromatographs, open the qualitative analysis program, and click file, open datafile.Select the file to be analyzed in the open data file window, and click open to open the file. Right click the mouse in the chromatogram results window and then extract chromatograms in a pop-up menu. In the type list, click on other chromatograms.In the detector combo box, select DAD1, then click okay to display the HPLC results in the chromatogram results window. Click the manual integration icon docked at the top of the chromatogram results window. Draw baseline for the peak required for manual integration analysis with the mouse.Click view, integration peak list, to display the results. Copy the results of the peak areas to Excel, and plot an HPLC based standard curve of the authentic flavonoid against the corresponding flavonoid concentrations, then calculate the yield of the flavonoid of interest produced in this protocol according the resulting formula. Then repeat the same procedures to extract single wavelength chromatograms to analyze the MS data for the exact mass of flavonoid compounds.Click the range select icon on the chromatogram results toolbar. Select the peak of interest. Right click the mouse in the selected range, and click the extract MS spectrum in the pop-up menu to display the results in the MS spectrum results window.To determine whether this in-vitro system can be used for the conversion of a flavanone into a flavonol, NRN was added into the system. There were two new spots emerged on a polyamide TLC plate. One spot showed a migration distance similar to that of DHK, and the others similar to that of KMF.Further analysis by HPLC and LC-MS demonstrated that the new chemical showed a retention time of 12 minutes and 20 minutes respectively, in a quasi-molecular ion peak M over Z at 287 and 285 which were identical to those of DHK and KMF. To determine whether the system can be used for the conversion of other flavanones into their corresponding flavonols, ERD was added into the system. Two new spots on a polyamide TLC plate displayed a migration distance similar to that of DHQ and QRC respectively.HPLC and LC-MS analyses demonstrated that these new chemicals revealed a retention time of 10 minutes and 16 minutes respectively, and a quasi-molecular ion peak M over Z at 303 and 301, which exactly corresponded to those of DHQ and QRC. When preparing the synthetic system, you should add the enzyme lastly, and add the ferrous sulfate second to last. Yes, it provides a guide for the economical production of other secondary metabolites.

biosynthesis-flavonol-from-flavanone-establishing-one-pot-bienzymatic

Research

JoVE Journal

Biochemistry

Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several target identification methods have been developed and used to successfully elucidate the binding proteins of a variety of small molecules, these techniques have drawbacks that make them unsuitable for detecting certain types of small molecule-target interactions. In particular, non-covalent interactions that depend on native cellular conditions, such as those of membrane proteins whose structures may be perturbed upon cell lysis, are often not amenable to affinity-based target identification methods. Here, we demonstrate a method wherein a probe containing a photolabile group is used to covalently crosslink to the small molecule binding protein within the environment of the live cell, allowing the detection and isolation of the target protein without the need for maintenance of the interaction after cell lysis. This technique is a valuable tool for studying biologically interesting small molecules with unknown mechanisms, both in the context of basic biology as well as drug discovery.

We describe here a method for identification of small molecule-binding proteins using photoaffinity labeling. The advantage of this technique is that binding and covalent labeling of the target proteins occurs within the live cellular environment, removing the risk of disrupting native protein structure and binding conditions upon cell lysis.

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several ...

Measuring Protein Binding to F-actin by Co-sedimentation

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, cross-linking and/or disassembly. This protocol describes a technique &#8211; the actin co-sedimentation, or pelleting, assay &#8211; to determine whether a protein or protein domain binds F-actin and to measure the affinity of the interaction (i.e., the dissociation equilibrium constant). In this technique, a protein of interest is first incubated with F-actin in solution. Then, differential centrifugation is used to sediment the actin filaments, and the pelleted material is analyzed by SDS-PAGE. If the protein of interest binds F-actin, it will co-sediment with the actin filaments. The products of the binding reaction (i.e., F-actin and the protein of interest) can be quantified to determine the affinity of the interaction. The actin pelleting assay is a straightforward technique for determining if a protein of interest binds F-actin and for assessing how changes to that protein, such as ligand binding, affect its interaction with F-actin.

This protocol describes a method to test the ability of a protein to co-sediment with filamentous actin (F-actin) and, if binding is observed, to measure the affinity of the interaction.

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, ...

Hydrolysis of a Ni-Schiff-Base Complex Using Conditions Suitable for Retention of Acid-labile Protecting Groups

Unnatural amino acids, amino acids containing side-chain functionalities not commonly seen in nature, are increasingly found in synthetic peptide sequences. Synthesis of some unnatural amino acids often includes the use of a precursor consisting of a Schiff-base stabilized by a nickel cation. Unnatural side-chains can be installed on an amino acid backbone found in this Schiff-base complex. The resulting unnatural amino acid can then be isolated from this complex using hydrolysis of the Schiff-base, typically by employing reflux in strongly acidic solution. These highly acidic conditions may remove acid-labile side-chain protecting groups necessary for the unnatural amino acids to be used in microwave-assisted solid-phase peptide synthesis. In this work, we present an efficient hydrolysis and subsequent Fmoc protection of an amino acid isolated from a Ni-Schiff base complex. Hydrolysis conditions presented in this work are suitable for retention of acid-labile side-chain protecting groups and may be adaptable to a variety of unnatural amino acid substrates.

Here, we present an efficient hydrolysis and subsequent Fmoc protection of an amino acid isolated from a Ni-Schiff-base complex. Hydrolysis conditions presented here are suitable for use when retention of acid-labile side-chain protecting groups is required. This technique may be adaptable to a variety of unnatural amino acid substrates.

Unnatural amino acids, amino acids containing side-chain functionalities not commonly seen in nature, are increasingly found in synthetic peptide ...

Protein Film Infrared Electrochemistry Demonstrated for Study of H2 Oxidation by a [NiFe] Hydrogenase

Understanding the chemistry of redox proteins demands methods that provide precise control over redox centers within the protein. The technique of protein film electrochemistry, in which a protein is immobilized on an electrode surface such that the electrode replaces physiological electron donors or acceptors, has provided functional insight into the redox reactions of a range of different proteins. Full chemical understanding requires electrochemical control to be combined with other techniques that can add additional structural and mechanistic insight. Here we demonstrate a technique, protein film infrared electrochemistry, which combines protein film electrochemistry with infrared spectroscopic sampling of redox proteins. The technique uses a multiple-reflection attenuated total reflectance geometry to probe a redox protein immobilized on a high surface area carbon black electrode. Incorporation of this electrode into a flow cell allows solution pH or solute concentrations to be changed during measurements. This is particularly powerful in addressing redox enzymes, where rapid catalytic turnover can be sustained and controlled at the electrode allowing spectroscopic observation of long-lived intermediate species in the catalytic mechanism. We demonstrate the technique with experiments on E. coli hydrogenase 1 under turnover (H2 oxidation) and non-turnover conditions.

Protein Film Infrared Electrochemistry Demonstrated for Study of H2 Oxidation by a [NiFe] Hydrogenase

Here, we describe a technique, protein film infrared electrochemistry, which allows immobilized redox proteins to be studied spectroscopically under direct electrochemical control at a carbon electrode. Infrared spectra of a single protein sample can be recorded at a range of applied potentials and under a variety of solution conditions.

Understanding the chemistry of redox proteins demands methods that provide precise control over redox centers within the protein. The technique of protein ...

A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the discovery of high-affinity interactors that are highly enriched in the library of interacting candidates. In a traditional format, the yeast 2-hybrid assay can yield too many colonies to analyze when conducted at low stringency where low affinity interactors might be found. Moreover, without a comprehensive and complete interrogation of the same library against different bait plasmids, a comparative analysis cannot be achieved. Although some of these problems can be addressed using arrayed prey libraries, the cost and infrastructure required to operate such screens can be prohibitive. As an alternative, we have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing a strategy termed DEEPN (Dynamic Enrichment for Evaluation of Protein Networks), which incorporates high-throughput DNA sequencing and computation to follow the evolution of a population of plasmids that encode interacting partners. Here, we describe customized reagents and protocols that allow a DEEPN screen to be executed easily and cost-effectively.

Batch processing of yeast 2-hybrid screens allows for direct comparison of the interaction profiles of multiple bait proteins with a highly complex set of prey fusion proteins. Here, we describe refined methods, new reagents, and how to implement their use for such screens.

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum (ER) resident proteins implicated in homotypic fusion of the ER. We detail here a method for purifying a glutathione S-transferase (GST) and poly-histidine tagged Drosophila atlastin by two rounds of affinity chromatography. Studying fusion reactions in vitro requires purified fusion proteins to be inserted into a lipid bilayer. Liposomes are ideal model membranes, as lipid composition and size may be adjusted. To this end, we describe a reconstitution method by detergent removal for Drosophila atlastin into preformed liposomes. While several reconstitution methods are available, reconstitution by detergent removal has several advantages that make it suitable for atlastins and other similar proteins. The advantage of this method includes a high reconstitution yield and correct orientation of the reconstituted protein. This method can be extended to other membrane proteins and for other applications that require proteoliposomes. Additionally, we describe a FRET based lipid mixing assay of proteoliposomes used as a measurement of membrane fusion.

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Biological membrane fusion is catalyzed by specialized fusion proteins. Measuring the fusogenic properties of proteins can be achieved by lipid mixing assays. We present a method for purifying recombinant Drosophila atlastin, a protein that mediates homotypic fusion of the ER, reconstituting it to preformed liposomes, and testing for fusion capacity.

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum ...

Obtaining High-Quality Transcriptome Data from Cereal Seeds by a Modified Method for Gene Expression Profiling

The characterization of gene expression is dependent on RNA quality. In germinating, developing and mature cereal seeds, the extraction of high-quality RNA is often hindered by high starch and sugar content. These compounds can reduce both the yield and the quality of the extracted total RNA. The deterioration in quantity and quality of total RNA can subsequently have a significant impact on the downstream transcriptomic analyses, which may not accurately reflect the spatial and/or temporal variation in the gene expression profile of the samples being tested. In this protocol, we describe an optimized method for extraction of total RNA with sufficient quantity and quality to be used for whole transcriptome analysis of cereal grains. The described method is suitable for several downstream applications used for transcriptomic profiling of developing, germinating, and mature cereal seeds. The method of transcriptome profiling using a microarray platform is shown. This method is specifically designed for gene expression profiling of cereals with described genome sequences. The detailed procedure from microarray handling to final quality control is described. This includes cDNA synthesis, cRNA labelling, microarray hybridization, slide scanning, feature extraction, and data quality validation. The data generated by this method can be used to characterize the transcriptome of cereals during germination, in various stages of grain development, or at different biotic or abiotic stress conditions. The results presented here exemplify high-quality transcriptome data amenable for downstream bioinformatics analyses, such as the determination of differentially expressed genes (DEGs), characterisation of gene regulatory networks, and conducting transcriptome-wide association study (TWAS).

A method for transcriptome profiling of cereals is presented. The microarray-based gene expression profiling starts with the isolation of high-quality total RNA from cereal grains and continues with the generation of cDNA. After cRNA labelling and microarray hybridization, recommendations are given for signal detection and quality control.

The characterization of gene expression is dependent on RNA quality. In germinating, developing and mature cereal seeds, the extraction of high-quality ...

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk population methods (Northern blot, PCR, and RNAseq) that measure mRNA levels lack the ability to provide information on cell-to-cell variation in responses. Precise single cell and allelic visualization and quantification is possible via single molecule RNA fluorescence in situ hybridization (smFISH). RNA-FISH is performed by hybridizing target RNAs with labeled oligonucleotide probes. These can be imaged in medium/high throughput modalities, and, through image analysis pipelines, provide quantitative data on both mature and nascent RNAs, all at the single cell level. The fixation, permeabilization, hybridization and imaging steps have been optimized in the lab over many years using the model system described herein, which results in successful and robust single cell analysis of smFISH labeling. The main goal with sample preparation and processing is to produce high quality images characterized by a high signal-to-noise ratio to reduce false positives and provide data that are more accurate. Here, we present a protocol describing the pipeline from sample preparation to data analysis in conjunction with suggestions and optimization steps to tailor to specific samples.&#160;

Single molecule RNA fluorescence in situ hybridization (smFISH) is a method to accurately quantify levels and localization of specific RNAs at the single cell level. Here, we report our validated lab protocols for wet-bench processing, imaging and image analysis for single cell quantification of specific RNAs.

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

Removal and Replacement of Endogenous Ligands from Lipid-Bound Proteins and Allergens

Many major allergens bind to hydrophobic lipid-like molecules, including Mus m 1, Bet v 1, Der p 2, and Fel d 1. These ligands are strongly retained and have the potential to influence the sensitization process either through directly stimulating the immune system or altering the biophysical properties of the allergenic protein. In order to control for these variables, techniques are required for the removal of endogenously bound ligands and, if necessary, replacement with lipids of known composition. The cockroach allergen Bla g 1 encloses a large hydrophobic cavity which binds a heterogeneous mixture of endogenous lipids when purified using traditional techniques. Here, we describe a method through which these lipids are removed using reverse-phase HPLC followed by thermal annealing to yield Bla g 1 in either its Apo-form or reloaded with a user-defined mixture of fatty acid or phospholipid cargoes. Coupling this protocol with biochemical assays reveal that fatty acid cargoes significantly alter the thermostability and proteolytic resistance of Bla g 1, with downstream implications for the rate of T-cell epitope generation and allergenicity. These results highlight the importance of lipid removal/reloading protocols such as the one described herein when studying allergens from both recombinant and natural sources. The protocol is generalizable to other allergen families including lipocalins (Mus m 1), PR-10 (Bet v 1), MD-2 (Der p 2) and Uteroglobin (Fel d 1), providing a valuable tool to study the role of lipids in the allergic response.

This protocol describes the removal of endogenous lipids from allergens, and their replacement with user-specified ligands through reverse-phase HPLC coupled with thermal annealing. 31P-NMR and circular dichroism allow for the rapid confirmation of ligand removal/loading, and the recovery of native allergen structure.

Many major allergens bind to hydrophobic lipid-like molecules, including Mus m 1, Bet v 1, Der p 2, and Fel d 1. These ligands are strongly retained and ...