Laurence, E. J. | bioRxiv Science

bioRxiv2025

Rapid Antibody Fragment Production and Binding Analysis Using Cell-Free Protein Synthesis Combined with Fluorescence Correlation Spectroscopy

This study investigates the efficient development and production of single-chain variable fragments (scFvs) and antibody fragments (Fabs) using an E. coli-based cell-free protein synthesis system. Validation of the methodology was performed using a fluorescence correlation spectroscopy (FCS)-based assay to determine binding equilibrium constants (KD) between antibodies and the receptor binding domain (RBD) of SARS-CoV-2 Spike protein. An initial assessment employed two conventionally cell-produced anti-RBD antibodies. To optimize cell-free production, folding strategies were developed to enhance the solubility and yields of scFvs, including a two-stage refolding protocol that successfully recovered active proteins from misfolded precipitates. Fab fragments were also produced and characterized, with their binding properties analyzed to assess functionality. This study highlights the potential of cell-free systems for the rapid and efficient production of functional antibody fragments. The integration of advanced techniques, such as FCS-based kinetic measurements, underscores the versatility and applicability of cell-free platforms for antibody development and high-throughput screening. These findings offer a promising avenue for accelerating therapeutic antibody research and production. SIGNIFICANCE of WORKThis study highlights the transformative potential of integrating cell-free protein synthesis (CFPS) with fluorescence correlation spectroscopy (FCS) for the rapid and scalable production and characterization of functional antibody fragments. By leveraging an E. coli-based CFPS platform, we successfully designed and optimized the production of single-chain variable fragments (scFvs) compared to Fab fragments, addressing some common challenges of low solubility and yield. The development of a cost-efficient two-stage refolding strategy further enhanced the scalability and functionality of scFvs, enabling higher recovery of active proteins from the unfolded state. Additionally, FCS provided a sensitive, rapid method for accurately quantifying antigen-antibody binding kinetics across a range of affinities. This work establishes CFPS and FCS as versatile and complementary tools, offering a robust framework for accelerating antibody fragment development, particularly in time-sensitive scenarios like infectious disease outbreaks.

biophysics↗

bioRxiv2024

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.

biophysics↗