A Cost-Effective Immobilization Method for MBP Fusion Proteins on Microtiter Plates Using a Gelatinized Starch–Agarose Mixture and Its Application for Convenient Protein–Protein Interaction Analysis

Abstract

The detection and quantification of protein–protein interactions (PPIs) is a crucial technique that often involves the use of recombinant proteins with fusion protein tags, such as maltose-binding protein (MBP) and glutathione-S-transferase (GST). In this study, we improved the cohesive and sticky properties of gelatinized starch by supplementing it with agarose, resulting in a harder gel that could coat the bottom of a microtiter plate. The resulting gelatinized starch/agarose mixture allowed for the efficient immobilization of MBP-tagged proteins on the coated plates, enabling the use of indirect ELISA-like PPI assays. By using the enzymatic activity of GST as an indicator, we succeeded in determining the dissociation constants between MBP-tagged and GST-tagged proteins on 96-well microtiter plates and a microplate reader without any expensive specialized equipment.

Keywords

• gelatinized starch
• maltose-binding protein
• microplate-based assay
• protein–protein interaction
• dissociation constant determination