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

Summary

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.

Abstract

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.

Introduction

Conventional pulldown is widely used to study protein-protein interactions¹. However, purified proteins often do not interact effectively in vitro^2,3, and some of them are insoluble without their binding partner^4,5. Such proteins might require co-translational assembly or the presence of molecular chaperones^5,6,7,8,9. Another limitation of conventional pulldown is the testing of possible heteromultimerization activity between domains that can exist as stable homo-oligomers assembled co-translationally^8,10, 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 problems^3,11. 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 module^15,16, 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.

Protocol

The schematic representation of the method workflow is shown in Figure 1.

1. Co-transformation of E. coli

1. 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.
   1. Grow bacteria of an appropriate strain in Luria-Bertani (LB) media at 37 °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.
   2. Centrifuge 1.0 mL of the bacterial suspension for 1 min at 9,000 x g at 4 °C, and discard the supernatant.
   3. 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.
   4. Centrifuge for 30 s at 8,000 x g at 4 °C, and discard the supernatant.
   5. Add 100 µL 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.
      ​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.
   6. Heat at 42 °C for 150 s, then chill for 1 min on ice.
   7. Add 1 mL of liquid LB media without antibiotics and incubate at 37 °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 °C.

2. Expression

1. 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 °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.
2. Grow the cells with a constant rotation of 220 rpm at 37 °C to an OD of 0.5-0.7, cool to room temperature (RT), and add isopropyl β-D-1-thiogalactopyranoside (IPTG) to 1 mM. Store a 20 µL aliquot of cell suspension as a control of the un-induced sample.
3. Incubate the cells with a constant rotation of 220 rpm at 18 °C overnight.
   NOTE: The optimal time and temperature of incubation may vary; 18 °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.
4. Divide the bacterial suspension into two parts (or more if more than two different tags were used) and store a 20 µL 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 °C for at least 6 months.

3. Pulldown assay

NOTE: The detailed procedures are described for proteins tagged with either 6xHis or MBP/GST. All procedures are performed at 4°C.

1. 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:
   1. 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).
   2. GST- or MBP-pulldown: Mix 20 mM Tris (pH 7.5 at 25 °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).
2. Disrupt the cells by sonication on ice. Store a 20 µl 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.
3. Centrifuge at 20,000 x g for 30 min. Collect 20 µL of the clarified lysate for subsequent SDS-PAGE analysis.
4. Equilibrate the resin (50 µL 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.
5. 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 µL of the unbound fraction for subsequent SDS-PAGE analysis.
6. 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:
   1. 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.
   2. GST- or MBP-pulldown: Mix 20 mM Tris (pH 7.5 at 25 °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.
7. 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.
8. 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.
9. Elute the bound proteins with 50 μL of the elution buffer in a shaker at 1,200 rpm for 10 min. The common recipes of elution buffers are:
   1. 6xHis-pulldown: Mix 30 mM HEPES (pH 7.5), 400 mM NaCl, 300 mM Imidazole, and 5 mM beta-mercaptoethanol.
   2. GST-pulldown: 20 mM Tris (pH 7.5 at 25 °C), 150 mM NaCl, 50 mM Glutathione (adjusted to pH 7.5 with basic Tris), and 5 mM DTT.
   3. MBP-pulldown: Mix 20 mM Tris (pH 7.5 at 25 °C), 150 mM NaCl, 40 mM maltose, and 5 mM DTT.
10. 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.

Results

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, ...

Discussion

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 experiment^3,11.

The ...

Materials

Name	Company	Catalog Number	Comments
8-ELEMENT probe	Sonics	630-0586	The high throughput 8-element sonicator probes
Agar	AppliChem	A0949
Amylose resin	New England Biolabs	E8021	Resin for purification of MBP-tagged proteins
Antibiotics	AppliChem	A4789 (kanamycin); A0839 (ampicillin)
Beta-mercaptoethanol	AppliChem	A1108
BL21(DE3)	Novagen	69450-M
CaCl2	AppliChem	A4689
Centrifuge	Eppendorf	5415R (Z605212)
Glutathione	AppliChem	A9782
Glutathione agarose	Pierce	16100	Resin for purification of GST-tagged proteins
Glycerol	AppliChem	A2926
HEPES	AppliChem	A3724
HisPur Ni-NTA Superflow Agarose	Thermo Scientific	25214	Resin for purification of 6xHis-tagged proteins
Imidazole	AppliChem	A1378
IPTG	AppliChem	A4773
KCl	AppliChem	A2939
LB	AppliChem	414753
Maltose	AppliChem	A3891
MOPS	AppliChem	A2947
NaCl	AppliChem	A2942
NP40	Roche	11754599001
pACYCDuet-1	Sigma-Aldrich	71147	Vector for co-expression of proteins with p15A replication origin
pCDFDuet-1	Sigma-Aldrich	71340	Vector for co-expression of proteins with CloDF13 replication origin
PMSF	AppliChem	A0999
Protease Inhibitor Cocktail VII	Calbiochem	539138	Protease Inhibitor Cocktail
pRSFDuet-1	Sigma-Aldrich	71341	Vector for co-expression of proteins with RSF replication origin
SDS	AppliChem	A2263
Tris	AppliChem	A2264
VC505 sonicator	Sonics	CV334	Ultrasonic liquid processor
ZnCl2	AppliChem	A6285