Expression and characterization of a new serine protease inhibitory protein in Escherichia coli

Hong, Tran Thi, et al. “Expression and Characterization of a New Serine Protease Inhibitory Protein in Escherichia Coli.” Biomedical Research and Therapy, vol. 7, no. 2, 2020, pp. 3633–3644., doi:10.15419/bmrat.v7i2.590.


Introduction: Proteases are enzymes that catalyze the hydrolysis of peptide bonds and play an important role in almost all biological processes. However, excessive protein proteolysis can be implicated in several diseases, such as cancer, as well as cardiovascular, inflammatory, neurodegenerative, bacterial, viral and parasitic diseases. In these cases, protease inhibitors can be used as one of versatile tools for regulating proteolytic activity of target proteases as well as therapeutic applications. In this study, we expressed and characterized a new serine protease inhibitory protein (PI-QT) from the metagenome of sponge-associated microorganisms in Escherichia coli.

Methods: The gene PI-QT encoding for a new serine protease inhibitory protein was expressed in E. coli BL21(DE3). In addition, the expressed protein was purified and characterized.

Results: Optimization of expression of the recombinant protein PI-QT in E. coli showed that suitable conditions for expression of the protein were pre-induction cell density (OD600) of 0.6 – 0.7, IPTG concentration of 1 mM and temperature of 25oC. The protease inhibitory protein was also purified and identified by mass spectrometry LC-MS/MS. The recombinant protein showed inhibitory activity against trypsin anda-chymotrypsin with activity values of 97526 U/mg and 41714 U/mg, respectively. Maximum activity of the protease inhibitory protein was obtained at pH 7 and temperature 20-35oC. The inhibitor was stable over pH 4-9 and up to temperature 50oC. Addition of Zn2+, Mg2+ and Ca2+ enhanced inhibitory activity, whereas other metal ions, surfactants and oxidants reduced inhibitory activity of the protease inhibitor.

Conclusion: The recombinant protein PI-QT is a potential protease inhibitor for therapeutic applications.