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.

Keywords: cell-free protein synthesis; cell-free synthetic biology; enzymatic activity; serratiopeptidase; therapeutic proteins.

Figure 1

Schematic diagram of cell-free protein synthesis (CFPS) and optimization for soluble and active expression of serratiopeptidase.

Figure 2

Optimization of the CFPS system for soluble expression of serratiopeptidase. (a) Western-blot analysis of cell-free expressed serratiopeptidase (50 kDa) using different cell extracts. The plasmid of pJL1-serratio with an N-terminal 6x His tag was used for CFPS reactions. (b) Western-blot analysis of cell-free expressed SUMO-serratiopeptidase (61 kDa) using different cell extracts. The plasmid of pET28a-SUMO-serratio with an N-terminal 6x His tag was used for CFPS reactions. In (a,b), CE: cell extracts prepared from E. coli BL21 Star (DE3); CE2: CE enriched with the chaperones of GroES-GroEL-tig; CE8: CE enriched with the chaperones of DnaK-DnaJ-GrpE and GroES-GroEL; NC: negative control without plasmid in the CFPS reaction; T: total protein; S: soluble protein; M: protein molecular weight marker. The protein band between 35 and 40 kDa was an unknown protein derived from the cell lysate.

Figure 3

Detection of the serratiopeptidase activity. (a) Confirming the activity of cell-free expressed serratiopeptidase. (b) Time course of the enzyme activity measured within 7 h incubation. One enzyme unit (U) is defined as an increase of 0.01 absorption (at 450 nm) unit after 30 min of enzymatic reaction at 45 °C. Values show means with error bars representing standard deviations (s.d.) of at least three independent experiments. Student’s t-tests were used for statistical analysis, and p < 0.05 indicated statistical significance (** p < 0.01).

Figure 4

Optimization of physicochemical factors for the assay of serratiopeptidase activity. Effects of (a) reaction pH, (b) reaction temperature, (c) azocasein concentration, and (d) metal ions on the serratiopeptidase activity. Note that each value was calculated by subtracting the background activity. Values show means with error bars representing standard deviations (s.d.) of at least three independent experiments.

Figure 5

Effects of lyophilization on the serratiopeptidase activity. Lyophilized-90: 90 μL of cell-free reaction mixture was freeze-dried in one tube; Lyophilized-180: 180 μL of cell-free reaction mixture was freeze-dried in one tube; Non-lyophilized: cell-free reaction mixture was not freeze-dried; Buffer: acetate buffer (pH 5.0); NFW: nuclease-free water. Values show means with error bars representing standard deviations (s.d.) of at least three independent experiments.