Recombinant Chitinase C produced through cell-free protein synthesis retains chitin-binding capabilities

Optimizing high-yield production of target proteins is often a time-consuming process for many experiments. Cell-free protein synthesis (CFPS) is a potential solution, as it eliminates preliminary processes, such as cell transformation, while providing high protein concentration. In this study, our target protein for CFPS production was Chitinase C (ChiC) from Pseudomonas aeruginosa PAO1. We hypothesized that ChiC would retain its chitin-binding functionality through CFPS synthesis. Our study demonstrated that CFPS can successfully produce recombinant ChiC in Escherichia coli BL21 (DE3) cell extracts that not only fold at the appropriate size, but also possess chitin-binding functionality. Overall, CFPS was revealed as a viable avenue for ChiC synthesis, laying the foundation for future optimization of ChiC production and cell-free production of similar proteins.