Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Summary

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

Abstract

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of K_D determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Introduction

Quantitative characterization of affinity of intermolecular interactions is important in many areas of biomedical research. Binding dissociation constant (K_D) is essential not only in drug discovery but is also an important parameter in characterization of any binary interaction in any biological system. Biochemical methods used for detection of protein-protein interactions, such as immunoprecipitation and yeast two-hybrid screens, do not inform us on how tight are those interactions, while affinity defines whether this particular complex exists under given conditions in vivo. In drug discovery process, binding assay development is one of the neces....

Protocol

1. Preparation of Cell Lysate

This protocol is intended for adherent cells expressing any GFP-fused protein. Needed cell number can vary from as low as 10⁶ to as many as 20 x 10⁶ cells, depending on the level of protein expression. For example, lysate of HEK cells overexpressing GFP-STAT3 was prepared by treating cells grown in 10 T75 flasks to near 70% confluency with 1 ml lysis buffer. However, this lysate had to be diluted 150-fold to provide optimal level of fluorescence for MST experiment. Cell lysis protocol depends strongly on the properties and intracellular localization of the protein under investigation. If usi....

Results

Measuring the affinity of non-phosphorylated STAT3 protein binding to oligonucleotides.

HEK293 cells expressing STAT3-GFP were used as a source of fluorescently labeled STAT3 for DNA binding assay. Cell lysates were prepared using RIPA buffer (20x10⁶cells/ml). For binding studies, the lysates were diluted 150x with MST DNA-binding buffer to provide the optimal level of the fluorescent protein in the binding reaction (about 20 nM). Non-transfected HEK293 cells have been.......

Discussion

Protein expression and purification is a laborious and expensive step, which is, however, necessary for determination of interactions' K_D by most currently used method. Application of MST allows avoiding protein purification thus significantly simplifying and accelerating quantitative characterization of interactions. It presents particularly significant advantages in the case of difficult-to-express and purify proteins, such as membrane proteins and transcription factors.

The major.......

Materials

Name	Company	Catalog Number	Comments
Name of the Reagent/material	Company	Catalogue Number	Comments
RIPA buffer	Millipore	20-188	Other manufacturer's buffers work as well
Protease inhibitors cocktail	Sigma-Aldrich	P2714
Monolith NT.115	NanoTemper Technologies GmbH	G008
Monolith NT.115 Capillary Tray	NanoTemper Technologies GmbH	T001
Monolith NT.115 Standard Treated Capillaries	NanoTemper Technologies GmbH	K002
NT Control software	NanoTemper Technologies GmbH	2.0.2.29
NT Analysis software	NanoTemper Technologies GmbH	1.4.27
Table-top refrigerated centrifuge	Eppendorf	5417R	Other microtube refrigerated centrifuges
Protein LoBind Tube 0.5 ml	Eppendorf	22431064