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 kanam...

<ol>
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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>
		</tr>
		<tr>
			<td>HEPES&#160;</td>
			<td>AppliChem</td>
			<td>A3724</td>
			<td></td>
		</tr>
		<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>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>AppliChem</td>
			<td>A4773</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>AppliChem</td>
			<td>A2939</td>
			<td></td>
		</tr>
		<tr>
			<td>LB</td>
			<td>AppliChem</td>
			<td>414753</td>
			<td></td>
		</tr>
		<tr>
			<td>Maltose</td>
			<td>AppliChem</td>
			<td>A3891</td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>AppliChem</td>
			<td>A2947</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>AppliChem</td>
			<td>A2942</td>
			<td></td>
		</tr>
		<tr>
			<td>NP40</td>
			<td>Roche</td>
			<td>11754599001</td>
			<td></td>
		</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>
		</tr>
		<tr>
			<td>pCDFDuet-1</td>
			<td>Sigma-Aldrich</td>
			<td>71340</td>
			<td>Vector for co-expression of proteins with CloDF13 replication origin</td>
		</tr>
		<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 ...

This method allows to study formations of protein complexes that require co-expression for efficient test samples, such as the formation of heterodimers. Main end potential of this method is exploiting the co-expression of differentially-tagged studied proteins using combinations of compatible vectors to promote efficient complex assembly, along with time-efficient workflow and scalability allowing rapid patch-testing of multiple interactions. This method can also be used for rapid screening of optimal protein expression and purification conditions.Visual demonstration helps correctly perform critical steps of the protocol such as co-expression setup, cell disruption, and subsequent pulldown steps. To begin, grow Escherichia coli BL21(DE3)strain in Luria-Bertani, or LB, media at 37 degrees Celsius to an optical density, or OD, of 0.1 to 0.2. Centrifuge one milliliter of the bacterial suspension for one minute at 9, 000 G at four degrees Celsius and discard the supernatant.Then, add 0.5 milliliters of ice-cold transformation buffer, or TB, and incubate for 10 minutes on ice. At the end of incubation, centrifuge for 30 seconds at 8, 000 G at four degrees Celsius and discard the supernatant. Then, add 100 microliters of TB buffer, 100 nanograms of each vector, and incubate for 30 minutes on ice.Heat at 42 degrees Celsius for 150 seconds, then chill on ice for one minute. Next, add one milliliter of LB media without antibiotics and incubate at 37 degrees Celsius for 90 minutes. After incubation, centrifuge and resuspend the pellet in 100 microliters of LB media before plating the suspension on an LB agar plate containing 0.5%glucose and corresponding antibiotics.Incubate the plate overnight at 37 degrees Celsius. The following day, flush the cells from the plate with two milliliters of LB media into 50 milliliters of LB media with corresponding antibiotics. Add metal ions or other known co-factors before storing an aliquot with 20%glycerol at minus 70 degrees Celsius for subsequent experiments.Grow the cells with a constant rotation of 220 RPM at 37 degrees Celsius to an OD of 0.5 to 0.7. Cool the culture to room temperature and add IPTG to a final concentration of one millimole. Store a 20-microliter aliquot of cell suspension as a control of the uninduced sample.Incubate the cells overnight at 220 RPM at 18 degrees Celsius. The next day, divide the bacterial suspension into two parts and store 20-microliter aliquots to confirm protein expression. Centrifuge the remaining cell suspension at 4, 000 G for 15 minutes.For pulldown assay, resuspend the pellets in one milliliter of ice-cold lysis buffer with protease inhibitors and reducing agents added immediately prior to the experiment. Adjust the buffer composition for the tested proteins. Disrupt the cells by sonication on ice and store a 20-microliter aliquot for electrophoresis.Centrifuge at 16, 000 G for 30 minutes and collect 20 microliters of the clarified lysate for electrophoresis. Equilibrate the resin with one milliliter of ice-cold lysis buffer for 10 minutes, then centrifuge at 2, 000 G for 30 seconds and discard the supernatant. Add cell lysates to the resin and incubate for 10 minutes at a constant rotation of 15 RPM.Then, centrifuge and discard the supernatant before collecting 20 microliters of the unbound fraction for analysis. Next, add one milliliter of ice-cold wash buffer and incubate for one minute. Again, centrifuge and discard the supernatant.Then, perform two long washes with ice-cold wash buffer. After the second wash, elute the bound proteins with 50 microliters of the elution buffer in a shaker at 1, 200 RPM at four degrees Celsius for 10 minutes. Analyze the eluted proteins with SDS polyacrylamide gel electrophoresis.Studies of the zinc finger-associated domain, or ZAD, homo-dimerization in maltose-binding protein, or MBP, and 6xHistidine pulldown assays are shown here. The co-expression of MBP and Thioredoxin 6xHistidine-fused ZADs shows good and reproducible homodimerization activity, appearing as an additional band in the SDS-PAGE results of the MBP pulldown assay. Another example of using the method to study alternative complex formation is shown here.In the co-expression assay, 6xHistidine-tagged ENY2 interacted both with GST-tagged Sgf11 and MBP-CTCF, but no GST-Sgf11 was present in the MBP pulldowns and vice-versa. A step-by-step comparison of required time intervals in the workflow of the conventional pulldown assay compared to pulldown coupled to co-expression is shown here. The results of utilizing both techniques to study the same interaction between the BTB domain of the CP190 protein and the GST-tagged C-terminal domain of Drosophila CTCF protein are shown here.The correct choice of affinity and solubility test, Thioredoxin, GST, or MBP, is critical for the efficient execution of the assay. Another critical step is fast and uniform cell disruption. It's highly recommended to use multi-tip sonicator probes.This method allow the direct study of the heterodimer formation between protein domains that otherwise form homodimers that would not disassociate during the incubation of purified proteins in conventional pulldown assay.

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

Research

JoVE Journal

Biochemistry

2.5K vistas.  Russian Academy of Sciences. Este método permite estudiar formaciones de complejos proteicos que requieren coexpresión para muestras de prueba eficientes, como la formación de heterodímeros. El principal potencial final de este método es explotar la coexpresión de proteínas estudiadas marcadas diferencialmente utilizando combinaciones de vectores compatibles para promover un ensamblaje complejo eficiente, junto con un flujo de trabajo eficiente y escalabilidad que permita pruebas rápidas de parches de múltiples ...