Real-time affinity measurements of proteins synthesized in cell-free lysate using fluorescence correlation spectroscopy

Rapid, high throughput measurements of biomolecular interactions are essential across medicine and bioscience. Traditional methods for affinity-screening proteins require a long and costly process involving cell-based expression, purification, and titration of multiple concentrations to arrive at a binding curve. In contrast, we have developed a fast and simple approach that yields a wealth of information about the expression of the protein and its binding characteristics, all in a "one-pot reaction" and done in under several hours without the need for protein purification. The method uses cell-free protein synthesis to produce the protein of interest in the presence of its binding partner, while simultaneously using fluorescence correlation spectroscopy (FCS) to measure the increasing concentration of the protein and its binding to the binding partner. We characterize the sensitivity limits of this method by measuring the binding between the green fluorescent protein (GFP) and a low picomolar-affinity anti-GFP antibody and found that we can quantify KDs down to the high picomolar to low-nanomolar range. We further demonstrate the method in a potentially ultrahigh-throughput sample format, in which FCS measurements are collected inside microcapsules. This work lays the foundation for a platform aimed at production and in situ affinity screening of thousands of different proteins.