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 pulldo....

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 ....

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

List of materials used in this article

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