Cell-Free Expression of a Therapeutic Protein Serratiopeptidase

Abstract

Serratiopeptidase is a clinical therapeutic protein for the treatment of human diseases such as arthritis, bronchitis, and thrombosis. Yet production of this protein in a heterologous host (e.g., Escherichia coli) is difficult due to the issue of protein insolubility and the requirement of laborious refolding procedures. Cell-free protein synthesis (CFPS) systems, derived from crude cell extracts, are effective platforms for the expression of recombinant proteins in vitro. Here, we report a new method to produce serratiopeptidase by using an E. coli-based CFPS system. After rational selection of cell extracts and construction of expression vectors, soluble expression of serratiopeptidase was achieved and the enzyme activity could be readily tested in the cell-free reaction mixture. By further optimizing the key parameters, optimum conditions for the enzyme activity assay were obtained, including the pH value at 5, reaction temperature at 45 °C, substrate concentration at 10 mg/mL, and supplementing Ca2+ ions at 5 mM. Moreover, the CFPS mixture was freeze-dried and the activity of serratiopeptidase could be regenerated by hydration without losing activity. Overall, the CFPS system enabled soluble expression of serratiopeptidase with catalytic activity, providing a new and promising approach for this enzyme production. Our work extends the utility of the cell-free platform to produce therapeutic proteins with clinical applications.

Functional Hydrogels for Delivery of the Proteolytic Enzyme Serratiopeptidase

“…Since protease enzymes have bivalent cations like zinc and calcium as essential co-factors within their structure, the loss in enzyme activity can be due to some competitive/noncompetitive interactions [ 30 ]. It is also known that calcium can protect the enzyme from autolysis, and by introducing additional amounts of Ca 2+ ions, the activity of SER can be increased [ 45 ]. Indeed, our experiments revealed that the enzyme activity can be increased by two times by adding Ca 2+ to the buffer (SER/CaCl 2 mass ratio1:1).…”

Expanding the toolbox of probiotic Escherichia coli Nissle 1917 for synthetic biology

“…CFPS systems have been widely developed for in vitro protein synthesis with the advantages of time‐saving, easily manipulable, microscale, high‐throughput, and portable. [ 48 ] CFPS has also inspired the multidiscipline research fields, including genetic engineering, [ 49 ] metabolic engineering, [ 50–53 ] high‐throughput screening, [ 54 ] pharmaceutical production, [ 55,56 ] and education. [ 57,58 ] Therefore, a powerful chassis is essential for establishment of CFPS system.…”

Expanding the toolbox of probioticEscherichia coliNissle 1917 for synthetic biology

“…CFPS systems have been widely developed for in vitro protein synthesis with the advantages of time-saving, easily manipulable, microscale, high-throughput, and portable. 45 CFPS has also inspired the multidiscipline research fields, including genetic engineering, 46 metabolic engineering, 47−50 high-throughput screening, 51 pharmaceutical production, 52,53 and education. 54,55 Therefore, a powerful chassis is essential for CFPS system establishment.…”