A novel microfluidics-based method for probing weak protein–protein interactions

Abstract

We report the use of a novel microfluidics-based method to detect weak protein-protein interactions between membrane proteins. The tight junction protein, claudin-2, synthesised in vitro using a cell-free expression system in the presence of polymer vesicles as membrane scaffolds, was used as a model membrane protein. Individual claudin-2 molecules interact weakly, although the cumulative effect of these interactions is significant. This effect results in a transient decrease of average vesicle dispersivity and reduction in transport speed of claudin-2-functionalised vesicles. Polymer vesicles functionalised with claudin-2 were perfused through a microfluidic channel and the time taken to traverse a defined distance within the channel was measured. Functionalised vesicles took 1.19 to 1.69 times longer to traverse this distance than unfunctionalised ones. Coating the channel walls with protein A and incubating the vesicles with anti-claudin-2 antibodies prior to perfusion resulted in the functionalised vesicles taking 1.75 to 2.5 times longer to traverse this distance compared to the controls. The data show that our system is able to detect weak as well as strong protein-protein interactions. This system offers researchers a portable, easily operated and customizable platform for the study of weak protein-protein interactions, particularly between membrane proteins.

Microfluidic approaches for the analysis of protein–protein interactions in solution

“…This method has been applied to study the self-assembly of oligomeric alpha-B crystallin (Challa et al 2018) and protein aggregation of lysozyme and silk fibroin (Toprakcioglu et al 2019). An additional example of labelfree PPI analysis utilised an electrochemical readout, to report a weak binding interaction between free and membranebound protein in a microfluidic diffusion-analysis assay (Tan et al 2012).…”