Streamlining Cell-Free Protein Synthesis Biosensors for Use in Human Fluids: in Situ RNase Inhibitor Production During Extract Preparation

Cell-free protein synthesis (CFPS) biosensors provide flexible, fast, and on-demand diagnosis of biomarkers in human body fluids. Considering the high concentrations of many different RNases in human body fluids, RNase inhibitor is an important reagent for the human-body-fluid-based biosensors and accounts for more than 90% of the total cost of E. coli-lysate-based CFPS biosensors. Here an E. coli extract with RNase inhibition activity was developed which reduces the total cost of a CFPS biosensor by ~90%. Murine RNase inhibitor (m-RI) was overexpressed in E. coli cells at different growth temperatures, redox buffers, and folding chaperones. Reduced growth temperature accompanied by overexpression of GroEL/ES folding chaperones were found to be necessary to maintain RNase inhibition activity despite reduced m-RI expression capability. Further, to optimize protein synthesis capability of CFPS biosensors, E. coli extract containing m-RI was mixed with regular E. coli extract that did not contain m-RI. The optimized CFPS biosensor reagent mixture was lyophilized and rehydrated with 0–100% (v/v) of three human body fluids of saliva, serum, and urine. Rehydration with saliva showed the least drop in protein expression compared to serum and urine. Finally, the CFPS biosensor reagents were assessed with a saliva-based CFPS biosensor sensitive to glutamine concentrations.