Single-Walled Carbon Nanotube Probes for Protease Characterization Directly in Cell-Free Expression Reactions

Proteins can be rapidly prototyped with cell-free expression (CFE) but in most cases there is a lack of probes or assays to measure their function directly in the cell lysate, thereby limiting the throughput of these screens. Increased throughput is needed to build standardized, sequence to function data sets to feed machine learning guided protein optimization. Herein, we describe the use of fluorescent single-walled carbon nanotubes (SWCNT) as effective probes for measuring protease activity directly in cell-free lysate. Substrate proteins were conjugated to carboxymethyl cellulose-wrapped SWCNT, yielding stable and sensitive probes for protease detection with a detection limit of 6.4 ng/mL for bacterial protease from <i>Streptomyces griseus</i>. These probes successfully measured subtilisin activity in unpurified CFE reactions, surpassing commercial assays. Furthermore, they enabled continuous monitoring of activity during synthesis of subtilisin in both purified and lysate-based CFE systems without compromising protein expression. Surface passivation techniques, such as preincubation with cell lysate and supplement components, reduced the initial signal loss and improved probe signal stability in the complex cell lysate environment. These modular probes can be used, as described, for high-throughput screening and optimization of proteases and, with the change of conjugated substrate, a wider range of other hydrolases.