This work was supported by the Russian Science Foundation projects 19-74-30026 (method development and validation) and 19-74-10099 (protein-protein interaction assays); and by the Ministry of Science and Higher Education of the Russian Federation-grant 075-15-2019-1661 (analysis of representative protein-protein interactions).

The schematic representation of the method workflow is shown in Figure 1.
1. Co-transformation of E. coli
<ol>
	<li>Prepare expression vectors for target proteins using standard cloning methods. 
	NOTE: Typically, a good starting point is to use conventional pGEX/pMAL vectors bearing an ampicillin resistance gene and ColE1 origin for the expression of GST/MBP-tagged proteins and a compatible vector with p15A or RSF origin and kanamycin resistance to express 6xHis-tagged proteins, in some cases combined with either Thioredoxin or SUMO-tag to increase solubility. Usually, several combinations of tags need to be tested prior to the experiment. The described method itself is convenient for batch testing the expression conditions of target proteins. It is important to note that most Rosetta strains already contain the plasmid with p15A origin for expressing the tRNAs for rare codon;, thus if using such strains is a possible option, the p15A plasmids should be avoided. See the Table of Materials for details.
	<ol>
		<li>Grow bacteria of an appropriate strain in Luria-Bertani (LB) media at 37 &#176;C to an optical density (OD) of 0.1-0.2. BL21(DE3) strain was used for examples in this study. 
		NOTE: It is recommended to use freshly prepared competent cells to achieve efficient co-transformation with two vectors. If more than two vectors need to be co-transformed, it is better to transform them sequentially to achieve good transformation efficiency. Electroporation is a good alternative.</li>
		<li>Centrifuge 1.0 mL of the bacterial suspension for 1 min at 9,000 x g at 4 &#176;C, and discard the supernatant.</li>
		<li>Add 0.5 mL of ice-cold buffer transformation buffer (TB) (10 mM MOPS [pH 6.7], 250 mM KCl, 55 mM MnCl2, and 15 mM CaCl2) and incubate for 10 min on ice.</li>
		<li>Centrifuge for 30 s at 8,000 x g at 4 &#176;C, and discard the supernatant.</li>
		<li>Add 100 &#181;L of TB buffer, add 100 ng of each vector, and incubate for 30 min on ice. Separately transform single vectors to study protein behavior without co-expression. Additionally, co-transform expression vectors in pairs with empty co-expression vectors for non-specific binding controls. 
		&#8203;NOTE: In the examples provided in this study, pGEX/pMAL vectors with corresponding cDNAs fused to GST/MBP cDNAs were used in combination with compatible pACYC-derived vectors bearing cDNA encoding partner protein domains fused to Thioredoxin cDNA.</li>
		<li>Heat at 42 &#176;C for 150 s, then chill for 1 min on ice.</li>
		<li>Add 1 mL of liquid LB media without antibiotics and incubate at 37 &#176;C for 90 min. Plate on LB-agar plates containing 0.5% glucose and corresponding antibiotics (common concentrations are: 50 mg/L ampicillin; 20 mg/L kanamycin; 50 mg/L streptomycin; 35 mg/L chloramphenicol). Incubate the plates overnight at 37 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Expression
<ol>
	<li>Flush the cells from the plate with 2 mL of liquid LB media into 50 mL of LB media with corresponding antibiotics (common concentrations are: 50mg/L ampicillin; 20 mg/L kanamycin; 50 mg/L streptomycin; 35 mg/L chloramphenicol). Add metal ions or other known co-factors (in the examples provided in this study, 0.2 mM ZnCl2 was added to the media). Store an aliquot with 20% glycerol at -70 &#176;C for a subsequent repeat of the experiment. 
	NOTE: It is recommended to flush several colonies directly from the plate to exclude the possibility of bad expression in a single isolated clone due to occasional recombination events between two plasmids.</li>
	<li>Grow the cells with a constant rotation of 220 rpm at 37 &#176;C to an OD of 0.5-0.7, cool to room temperature (RT), and add isopropyl &#946;-D-1-thiogalactopyranoside (IPTG) to 1 mM. Store a 20 &#181;L aliquot of cell suspension as a control of the un-induced sample.</li>
	<li>Incubate the cells with a constant rotation of 220 rpm at 18 &#176;C overnight. 
	NOTE: The optimal time and temperature of incubation may vary; 18 &#176;C overnight works best for most proteins and is advised to be tried by default. Reduce the incubation time to 2-3 h if a strong non-specific binding is observed.</li>
	<li>Divide the bacterial suspension into two parts (or more if more than two different tags were used) and store a 20 &#181;L aliquot of the cell suspension to confirm protein expression. Centrifuge at 4,000 x g for 15 min. 
	NOTE: Pause point: bacterial pellets can be stored at -70 &#176;C for at least 6 months.</li>
</ol>
3. Pulldown assay
NOTE: The detailed procedures are described for proteins tagged with either 6xHis or MBP/GST. All procedures are performed at 4&#176;C.
<ol>
	<li>Resuspend the bacterial pellets in 1 mL of ice-cold lysis buffer with protease inhibitors and reducing agents (see below) added immediately prior to the experiment. Avoid dithiotreitol (DTT) when using metal-chelating resins since it strips out metal ions. Adjust the buffer composition for the tested proteins. The common recipes of lysis buffers found suitable for most proteins are:
	<ol>
		<li>6xHis-pulldown: Mix 30 mM HEPES (pH 7.5), 400 mM NaCl, 10 mM Imidazole, 0.1% NP40, 10% [w/w] glycerol, 5 mM beta-mercaptoethanol, 1 mM phenylmethylsfulfonylfluoride (PMSF), and a 1:1,000 dilution of the protease inhibitor cocktail (see Table of Materials).</li>
		<li>GST- or MBP-pulldown: Mix 20 mM Tris (pH 7.5 at 25 &#176;C), 150 mM NaCl, 10 mM KCl, 10 mM MgCl2, 0.1 mM ZnCl2, 0.1% NP40, 10% [w/w] glycerol, 5 mM DTT, 1 mM PMSF, and a 1:1,000 dilution of the protease inhibitor cocktail (see Table of Materials).</li>
	</ol>
	</li>
	<li>Disrupt the cells by sonication on ice. Store a 20 &#181;l aliquot for electrophoresis. 
	NOTE: Typically, 20-25 pulses of 5 s with 15 s intervals with 20 W output power are required per sample. The appropriate sonication power should be adjusted for each instrument to avoid overheating and ensure total cell disruption. To achieve better performance, it is strongly recommended to use high-throughput multi-tip sonicator probes.</li>
	<li>Centrifuge at 20,000 x g for 30 min. Collect 20 &#181;L of the clarified lysate for subsequent SDS-PAGE analysis.</li>
	<li>Equilibrate the resin (50 &#181;L for each sample) with 1 mL of ice-cold lysis buffer for 10 min, centrifuge at 2,000 x g for 30 s, and discard the supernatant.</li>
	<li>Add cell lysates (total protein concentration: 20-50 mg/mL) to the resin, incubate for 10 min at a constant rotation of 15 rpm, centrifuge at 2,000 x g for 30 s, and discard the supernatant. Collect 20 &#181;L of the unbound fraction for subsequent SDS-PAGE analysis.</li>
	<li>Add 1 mL of ice-cold wash buffer and incubate for 1 min. Centrifuge at 2,000 x g for 30 s, and discard the supernatant. The common recipes for wash buffers are:
	<ol>
		<li>6xHis-pulldown: Mix 30 mM HEPES (pH 7.5), 400 mM NaCl, 30 mM Imidazole, 0.1% NP40, 10% [w/w] glycerol, and 5 mM beta-mercaptoethanol.</li>
		<li>GST- or MBP-pulldown: Mix 20 mM Tris (pH 7.5 at 25 &#176;C), 500 mM NaCl, 10 mM KCl, 10 mM MgCl2, 0.1 mM ZnCl2, 0.1% NP40, 10% [w/w] glycerol, and 5 mM DTT.</li>
	</ol>
	</li>
	<li>Perform two long washes: add 1 mL of ice-cold wash buffer, incubate for 10-30 min with a constant rotation of 15 rpm, centrifuge at 2,000 x g for 30 s, and discard the supernatant.</li>
	<li>Add 1 mL of ice-cold wash buffer, incubate for 1 min, centrifuge at 2,000 x g for 30 s, and discard the supernatant.</li>
	<li>Elute the bound proteins with 50 &#956;L of the elution buffer in a shaker at 1,200 rpm for 10 min. The common recipes of elution buffers are:
	<ol>
		<li>6xHis-pulldown: Mix 30 mM HEPES (pH 7.5), 400 mM NaCl, 300 mM Imidazole, and 5 mM beta-mercaptoethanol.</li>
		<li>GST-pulldown: 20 mM Tris (pH 7.5 at 25 &#176;C), 150 mM NaCl, 50 mM Glutathione (adjusted to pH 7.5 with basic Tris), and 5 mM DTT.</li>
		<li>MBP-pulldown: Mix 20 mM Tris (pH 7.5 at 25 &#176;C), 150 mM NaCl, 40 mM maltose, and 5 mM DTT.</li>
	</ol>
	</li>
	<li>Analyze the eluted proteins with SDS-PAGE. 
	NOTE: The percentage of acrylamide should be adjusted to the size of the proteins. In the examples provided in this study, 12% acrylamide gels were used, running in tris-glycine-SDS buffer (2 mM Tris, 250 mM Glycine, 0.1% SDS) at constant voltage of 180 V. Gels were stained by boiling in 0.2% Coomassie blue R250, 10% acetic acid, and 30% isopropanol, and de-stained by boiling in 10% acetic acid. The amount of loaded protein should not be equal, since various amounts of interacting proteins can be pulled down in different experiments.</li>
</ol>

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The authors declare no competing interests.

The described method allows the testing of protein-protein interactions that cannot be efficiently assembled in vitro and require co-expression. The method is one of the few suitable approaches for studying heterodimerizing proteins, which are also capable of homodimerization since, when purified separately, such proteins form stable homodimers which most often cannot dissociate and re-associate during the experiment3,11.
The ...

Conventional pulldown is widely used to study protein-protein interactions1. However, purified proteins often do not interact effectively in vitro2,3, and some of them are insoluble without their binding partner4,5. Such proteins might require co-translational assembly or the presence of molecular chaperones5,6,7,8,9. Another limitation of conventional pulldown is the testing of possible heteromultimerization activity between domains that can exist as stable homo-oligomers assembled co-translationally8,10, as many of them cannot dissociate and re-associate in vitro during the incubation time. Co-expression was found to be useful in overcoming such problems3,11. Co-expression using compatible vectors in bacteria was successfully used to purify large multi-subunit macromolecular complexes, inclduing polycomb repressive complex PRC212, RNA polymerase II mediator head module13, bacteriophage T4 baseplate14, SAGA complex deubiquitinylase module15,16, and ferritin17. Replication origins commonly used for co-expression are ColE1, p15A18, CloDF1319, and RSF20. In the commercially available Duet expression system, these origins are combined with different antibiotic resistance genes and convenient multiple cloning sites to produce polycistronic vectors, allowing the expression of up to eight proteins. These origins have different copy numbers and can be used in varying combinations to achieve balanced expression levels of target proteins21. To test protein-protein interactions, various affinity tags are used; the most common are 6xHistidine, glutathione-S-transferase (GST), and maltose-binding protein (MBP), each of which has a specific affinity to the corresponding resin. GST and MBP also enhance the solubility and stability of tagged proteins22.
A number of methods involving protein co-expression in eukaryotic cells have also been developed, the most prominent of which is yeast two-hybrid assay (Y2H)23. Y2H assay is cheap, easy, and allows the testing of multiple interactions; however, its workflow takes more than 1 week to complete. There are also a few less frequently used mammalian cell-based assays, for example, fluorescent two-hybrid assay (F2H)24 and cell array protein-protein interaction assay (CAPPIA)25. F2H assay is relatively fast, allowing to observe protein interactions in their native cellular environment, but involves using expensive imaging equipment. All these methods have an advantage over prokaryotic expression providing the native eukaryotic translation and folding environment; however, they detect interaction indirectly, either by transcriptional activation or by fluorescent energy transfer, which often produces artifacts. Also, eukaryotic cells may contain other interaction partners of proteins of interest, which can interfere with the testing of binary interactions between proteins of higher eukaryotes.
The present study describes a method for the bacterial co-expression of differentially tagged proteins followed by conventional pulldown techniques. The method allows studying interactions between target proteins that require co-expression. It is more time-efficient compared to traditional pulldown, allowing batch testing of multiple targets, which makes it advantageous in most cases. Co-expression using compatible vectors is more convenient than polycistronic co-expression since it does not require a laborious cloning step.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>8-ELEMENT probe</td>
			<td>Sonics</td>
			<td>630-0586</td>
			<td>The high throughput 8-element sonicator probes</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>AppliChem</td>
			<td>A0949</td>
			<td></td>
		</tr>
		<tr>
			<td>Amylose resin</td>
			<td>New England Biolabs</td>
			<td>E8021</td>
			<td>Resin for purification of MBP-tagged proteins</td>
		</tr>
		<tr>
			<td>Antibiotics</td>
			<td>AppliChem</td>
			<td>A4789 (kanamycin); A0839 (ampicillin)</td>
			<td></td>
		</tr>
		<tr>
			<td>Beta-mercaptoethanol</td>
			<td>AppliChem</td>
			<td>A1108</td>
			<td></td>
		</tr>
		<tr>
			<td>BL21(DE3)&#160;</td>
			<td>Novagen</td>
			<td>69450-M</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>AppliChem</td>
			<td>A4689</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5415R (Z605212)</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutathione</td>
			<td>AppliChem</td>
			<td>A9782</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutathione agarose</td>
			<td>Pierce</td>
			<td>16100</td>
			<td>Resin for purification of GST-tagged proteins</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>AppliChem</td>
			<td>A2926</td>
			<td></td>
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			<td>HEPES&#160;</td>
			<td>AppliChem</td>
			<td>A3724</td>
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		<tr>
			<td>HisPur Ni-NTA Superflow Agarose</td>
			<td>Thermo Scientific</td>
			<td>25214</td>
			<td>Resin for purification of 6xHis-tagged proteins</td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>AppliChem</td>
			<td>A1378</td>
			<td></td>
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			<td>IPTG</td>
			<td>AppliChem</td>
			<td>A4773</td>
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		<tr>
			<td>KCl</td>
			<td>AppliChem</td>
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			<td>AppliChem</td>
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			<td>MOPS</td>
			<td>AppliChem</td>
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			<td>NaCl</td>
			<td>AppliChem</td>
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		<tr>
			<td>NP40</td>
			<td>Roche</td>
			<td>11754599001</td>
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		</tr>
		<tr>
			<td>pACYCDuet-1</td>
			<td>Sigma-Aldrich</td>
			<td>71147</td>
			<td>Vector for co-expression of proteins with p15A replication origin</td>
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		<tr>
			<td>pCDFDuet-1</td>
			<td>Sigma-Aldrich</td>
			<td>71340</td>
			<td>Vector for co-expression of proteins with CloDF13 replication origin</td>
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		<tr>
			<td>PMSF</td>
			<td>AppliChem</td>
			<td>A0999</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail VII</td>
			<td>Calbiochem</td>
			<td>539138</td>
			<td>Protease Inhibitor Cocktail</td>
		</tr>
		<tr>
			<td>pRSFDuet-1</td>
			<td>Sigma-Aldrich</td>
			<td>71341</td>
			<td>Vector for co-expression of proteins with RSF replication origin</td>
		</tr>
		<tr>
			<td>SDS&#160;</td>
			<td>AppliChem</td>
			<td>A2263</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris&#160;</td>
			<td>AppliChem</td>
			<td>A2264</td>
			<td></td>
		</tr>
		<tr>
			<td>VC505 sonicator</td>
			<td>Sonics</td>
			<td>CV334</td>
			<td>Ultrasonic liquid processor</td>
		</tr>
		<tr>
			<td>ZnCl2</td>
			<td>AppliChem</td>
			<td>A6285</td>
			<td></td>
		</tr>
	</tbody>
</table>

pulldown assay coupled with co-expression in bacteria cells as a time-efficient tool for testing challenging protein-protein interactions

Pulldown is an easy and widely used protein-protein interaction assay. However, it has limitations in studying protein complexes that do not assemble effectively in vitro. Such complexes may require co-translational assembly and the presence of molecular chaperones; either they form stable oligomers which cannot dissociate and re-associate in vitro or are unstable without a binding partner. To overcome these problems, it is possible to use a method based on the bacterial co-expression of differentially tagged proteins using a set of compatible vectors followed by the conventional pulldown techniques. The workflow is more time-efficient compared to traditional pulldown because it lacks the time-consuming steps of separate purification of interacting proteins and their following incubation. Another advantage is a higher reproducibility due to a significantly smaller number of steps and a shorter period of time in which proteins that exist within the in vitro environment are exposed to proteolysis and oxidation. The method was successfully applied for studying a number of protein-protein interactions when other in vitro techniques were found to be unsuitable. The method can be used for batch testing protein-protein interactions. Representative results are shown for studies of interactions between BTB domain and intrinsically disordered proteins, and of heterodimers of zinc-finger-associated domains.

The described method was used routinely with many different targets. Presented here are some representative results which likely cannot be obtained using conventional pulldown techniques. The first is the study of specific ZAD (Zinc-finger-associated domain) dimerization11. ZADs form stable and specific dimers, with heterodimers possible only between closely related domains within paralogous groups. The dimers formed by these domains are stable and do not dissociate for at least a few days; thus, ...

Watch this Scientific Journal Video about Pulldown Assay Coupled with Co-Expression in Bacteria Cells as a Time-Efficient Tool for Testing Challenging Protein-Protein Interactions at JoVE.com

Pulldown Assay Coupled with Co-Expression in Bacteria Cells as a Time-Efficient Tool for Testing Challenging Protein-Protein Interactions

Here, we describe a method for the bacterial co-expression of differentially tagged proteins using a set of compatible vectors, followed by the conventional pulldown techniques to study protein complexes that cannot assemble in vitro.

Pulldown is an easy and widely used protein-protein interaction assay. However, it has limitations in studying protein complexes that do not assemble ...

pulldown-assay-coupled-with-co-expression-bacteria-cells-as-time

Research

JoVE Journal

Biochemistry

2.7K Views.  Russian Academy of Sciences. Here, we describe a method for the bacterial co-expression of differentially tagged proteins using a set of compatible vectors, followed by the conventional pulldown techniques to study protein complexes that cannot assemble in vitro.

Video: Pulldown Assay Coupled with Co-Expression in Bacteria Cells as a Time-Efficient Tool for Testing Challenging Protein-Protein Interactions

Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is necessary to characterize it at the structural and mechanistic level. Several biochemical and biophysical methods exist for such purpose. Among them, fluorescence anisotropy is a powerful technique particularly used when the fluorescence intensity of a fluorophore-labeled protein remains constant upon protein-protein interaction. In this technique, a fluorophore-labeled protein is excited with vertically polarized light of an appropriate wavelength that selectively excites a subset of the fluorophores according to their relative orientation with the incoming beam. The resulting emission also has a directionality whose relationship in the vertical and horizontal planes defines anisotropy (r) as follows: r=(IVV-IVH)/(IVV+2IVH), where IVV and IVH are the fluorescence intensities of the vertical and horizontal components, respectively. Fluorescence anisotropy is sensitive to the rotational diffusion of a fluorophore, namely the apparent molecular size of a fluorophore attached to a protein, which is altered upon protein-protein interaction. In the present text, the use of fluorescence anisotropy as a tool to study protein-protein interactions was exemplified to address the binding between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor like-1 GTPase (EFL1). Conventionally, labeling of a protein with a fluorophore is carried out on the thiol groups (cysteine) or in the amino groups (the N-terminal amine or lysine) of the protein. However, SBDS possesses several cysteines and lysines that did not allow site directed labeling of it. As an alternative technique, the dye 4&#39;,5&#39;-bis(1,3,2 dithioarsolan-2-yl) fluorescein was used to specifically label a tetracysteine motif, Cys-Cys-Pro-Gly-Cys-Cys, genetically engineered in the C-terminus of the recombinant SBDS protein. Fitting of the experimental data provided quantitative and mechanistic information on the binding mode between these proteins.

Protein interactions are at the heart of a cell&#39;s function. Calorimetric and spectroscopic techniques are commonly used to characterize them. Here we describe fluorescence anisotropy as a tool to study the interaction between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor-like 1 GTPase (EFL1).

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is ...

Utilizing the Ethylene-releasing Compound, 2-Chloroethylphosphonic Acid, as a Tool to Study Ethylene Response in Bacteria

Ethylene (C2H4) is a gaseous phytohormone that is involved in numerous aspects of plant development, playing a dominant role in senescence and fruit ripening. Exogenous ethylene applied during early plant development triggers the triple response phenotype; a shorter and thicker hypocotyl with an exaggerated apical hook. Despite the intimate relationship between plants and bacteria, the effect of exogenous ethylene on bacteria has been greatly overlooked. This is partly due to the difficulty of controlling gaseous ethylene within the laboratory without specialized equipment. 2-Chloroethylphosphonic acid (CEPA) is a compound that decomposes into ethylene, chlorine, and phosphate in a 1:1:1:1 molar ratio when dissolved in an aqueous medium of pH 3.5 or greater. Here we describe the use of CEPA to produce in situ ethylene for the investigation of ethylene response in bacteria using the fruit-associated, cellulose-producing bacterium Komagataeibacter xylinus as a model organism. The protocols described herein include both the verification of ethylene production from CEPA via the Arabidopsis thaliana triple response assay and the effects of exogenous ethylene on K. xylinus cellulose production, pellicle properties and colonial morphology. These protocols can be adapted to examine the effect of ethylene on other microbes using appropriate growth media and phenotype analyses. The use of CEPA provides researchers with a simple and efficient alternative to pure ethylene gas for the routine determination of bacterial ethylene response.

The protocols outlined herein facilitate the convenient investigation of bacterial ethylene responses by utilizing 2-chloroethylphosphonic acid (CEPA). Ethylene is produced in situ through the decomposition of CEPA in an aqueous bacterial growth medium, circumventing the requirement for pure ethylene gas.

Ethylene (C2H4) is a gaseous phytohormone that is involved in numerous aspects of plant development, playing a dominant role in senescence and fruit ...

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The modification presented includes the combination of this technique with cell-penetrating sequences. We propose to design cell-penetrating baits that can be incubated with living cells instead of cell lysates and therefore the interactions found will reflect those that occur within the intracellular context. Connexin43 (Cx43), a protein that forms gap junction channels and hemichannels is down-regulated in high-grade gliomas. The Cx43 region comprising amino acids 266-283 is responsible for the inhibition of the oncogenic activity of c-Src in glioma cells. Here we use TAT as the cell-penetrating sequence, biotin as the pull-down tag and the region of Cx43 comprised between amino acids 266-283 as the target to find intracellular interactions in the hard-to-transfect human glioma stem cells. One of the limitations of the proposed method is that the molecule used as bait could fail to fold properly and, consequently, the interactions found could not be associated with the effect. However, this method can be especially interesting for the interactions involved in signal transduction pathways because they are usually carried out by intrinsically disordered regions and, therefore, they do not require an ordered folding. In addition, one of the advantages of the proposed method is that the relevance of each residue on the interaction can be easily studied. This is a modular system; therefore, other cell-penetrating sequences, other tags, and other intracellular targets can be employed. Finally, the scope of this protocol is far beyond protein-protein interaction because this system can be applied to other bioactive cargoes such as RNA sequences, nanoparticles, viruses or any molecule that can be transduced with cell-penetrating sequences and fused to pull-down tags to study their intracellular mechanism of action.

This is a protocol to study intracellular protein-protein interactions based on the biotin-avidin pull-down system with the novelty of combining cell-penetrating sequences. The main advantage is that the target sequence is incubated with living cells instead of cell lysates and therefore the interactions will occur within the cellular context.

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA is a novel transcription activator found specifically in the obligate intracellular bacterial pathogen Chlamydia. Protein pulldown assays using affinity beads have revealed that GrgA binds two &#963; factors, namely &#963;66 and &#963;28, which recognize different sets of promoters for genes whose products are differentially required at developmental stages. We have used BLI to confirm and further characterize the interactions. BLI demonstrates several advantages over pulldown: 1) It reveals real-time association and dissociation between binding partners, 2) It generates quantitative kinetic parameters, and 3) It can detect bindings that pulldown assays often fail to detect. These characteristics have enabled us to deduce the physiological roles of GrgA in gene expression regulation in Chlamydia, and possible detailed interaction mechanisms. We envision that this relatively affordable technology can be extremely useful for studying transcription and other biological processes.

Application of Biolayer Interferometry BLI for Studying Protein-Protein Interactions in Transcription

Interactions of transcription factors (TFs) with the RNA polymerase are usually studied using pulldown assays. We apply a Biolayer Interferometry (BLI) technology to characterize the interaction of GrgA with the chlamydial RNA polymerase. Compared to pulldown assays, BLI detects real-time association and dissociation, offers higher sensitivity, and is highly quantitative.

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA ...

Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions largely depend on intracellular protein&#8211;protein interactions. Therefore, research regarding these interactions is indispensable to facilitating the understanding of physiologic processes. Co-precipitation of associated proteins, followed by mass spectrometry (MS) analysis, enables the identification of novel protein interactions. In this study, we have provided details of the novel technique of immunoprecipitation-liquid chromatography (LC)-MS/MS analysis combined with on-membrane digestion for the analysis of protein&#8211;protein interactions. This technique is suitable for crude immunoprecipitants and can improve the throughput of proteomic analyses. Tagged recombinant proteins were precipitated using specific antibodies; next, immunoprecipitants blotted onto polyvinylidene difluoride membrane pieces were subjected to reductive alkylation. Following trypsinization, the digested protein residues were analyzed using LC-MS/MS. Using this technique, we were able to identify several candidate associated proteins. Thus, this method is convenient and useful for the characterization of novel protein&#8211;protein interactions.

Here, we present a protocol for an on-membrane digestion technique for the preparation of samples for mass spectrometry. This technique facilitates the convenient analysis of protein&#8211;protein interactions.

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions ...

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary mechanism of amyloid aggregate-induced toxicity in neurodegenerative diseases. However, most reports describing the mechanisms of membrane disruption are based on phospholipid model systems, and studies directly targeting events occurring at the level of biological membranes are rare. Described here is a model for studying the mechanisms of amyloid toxicity at the membrane level. For mitochondrial isolation, density gradient medium is used to obtain preparations with minimal myelin contamination. After mitochondrial membrane integrity confirmation, the interaction of amyloid fibrils arising from &#945;-synuclein, bovine insulin, and hen egg white lysozyme (HEWL) with rat brain mitochondria, as an in vitro biological model, is investigated. The results demonstrate that treatment of brain mitochondria with fibrillar assemblies can cause different degrees of membrane permeabilization and ROS content enhancement. This indicates structure-dependent interactions between amyloid fibrils and mitochondrial membrane. It is suggested that biophysical properties of amyloid fibrils and their specific binding to mitochondrial membranes may provide explanations for some of these observations.

Provided here is a protocol for investigating the interactions between native form, prefibrillar, and mature amyloid fibrils of different peptides and proteins with mitochondria isolated from different tissues and various areas of the brain.

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary ...

MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria

Although small regulatory RNAs (sRNAs) are widespread among the bacterial domain of life, the functions of many of them remain poorly characterized notably due to the difficulty of identifying their mRNA targets. Here, we described a modified protocol of the MS2-Affinity Purification coupled with RNA Sequencing (MAPS) technology, aiming to reveal all RNA partners of a specific sRNA in vivo. Broadly, the MS2 aptamer is fused to the 5&#8217; extremity of the sRNA of interest. This construct is then expressed in vivo, allowing the MS2-sRNA to interact with its cellular partners. After bacterial harvesting, cells are mechanically lysed. The crude extract is loaded into an amylose-based chromatography column previously coated with the MS2 protein fused to the maltose binding protein. This enables the specific capture of MS2-sRNA and interacting RNAs. After elution, co-purified RNAs are identified by high-throughput RNA sequencing and subsequent bioinformatic analysis. The following protocol has been implemented in the Gram-positive human pathogen Staphylococcus aureus and is, in principle, transposable to any Gram-positive bacteria. To sum up, MAPS technology constitutes an efficient method to deeply explore the regulatory network of a particular sRNA, offering a snapshot of its whole targetome. However, it is important to keep in mind that putative targets identified by MAPS still need to be validated by complementary experimental approaches.

MAPS technology has been developed to scrutinize the targetome of a specific regulatory RNA in vivo. The sRNA of interest is tagged with a MS2 aptamer enabling the co-purification of its RNA partners and their identification by RNA sequencing. This modified protocol is particularly suited for Gram-positive bacteria.&#160;

Although small regulatory RNAs (sRNAs) are widespread among the bacterial domain of life, the functions of many of them remain poorly characterized ...

Pulldown Assay Coupled with Co-Expression in Bacteria Cells as a Time-Efficient Tool for Testing Challenging Protein-Protein Interactions

Summary

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Chapters in this video

Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions

Utilizing the Ethylene-releasing Compound, 2-Chloroethylphosphonic Acid, as a Tool to Study Ethylene Response in Bacteria

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

Evaluation of Protein–Protein Interactions using an On-Membrane Digestion Technique

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria