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Characterization of a metalloprotease inhibitor protein (SmaPI) of Serratia marcescens
1Department of Microbiology, College of Natural Sciences, Gyeongsang National University, Chinju, Republic of Korea.
Abstract:
As suggested by Y. Suh and M.J. Benedik (J. Bacteriol. 174: 2361-2366, 1992), Serratia marcescens ATCC 27117 produced very small amounts (0.8 U ml-1) of an inhibitor protein (SmaPI) that shows an inhibitory activity against extracellular 50-kDa metalloprotease (SMP) of S. marcescens and that is localized in the periplasm of cells at the optimal growth temperature of 25 degrees C. A recombinant S. marcescens harboring plasmid pSP2 encoding SMP and SmaPI genes produced 20 U of SmaPI ml-1 that is also localized in the periplasm of cells at 25 degrees C. However, a large amount of SmaPI (86 Uml-1) was extracellularly produced at the supraoptimal growth temperature 37 degrees C from the recombinant S. marcescens (pSP2). We purified SmaPI from the culture supernatant of S. marcescens (pSP2) grown at 37 degrees C, and some biochemical properties were characterized. SmaPI had a pI value of about 10.0 and was a monomeric protein with a molecular mass of 10,000. SmaPI was produced from a precursor SmaPI by cleavage of a signal peptide (26 amino acid residues). The inhibitor was stable in boiling water for up to 30 min. The thermostability of SmaPI can be attributed to its reversible denaturation. SmaPI inhibited SMP by formation of a noncovalent complex with a molar ratio of 1:1 and showed a high protease specificity, which inhibited only SMP among the various proteases we examined.
Insights
Serratia marcescens produces a thermostable inhibitor protein (SmaPI) that specifically targets extracellular metalloprotease (SMP). Production increases significantly at higher temperatures, leading to extracellular release and purification of SmaPI.
Area of Science:
- Microbiology
- Protein Biochemistry
Background:
- Serratia marcescens produces an inhibitor protein (SmaPI) targeting its extracellular metalloprotease (SMP).
- SmaPI is typically periplasmically localized at optimal growth temperatures (25°C).
Purpose of the Study:
- To investigate the effect of supraoptimal temperature (37°C) on SmaPI production and localization.
- To characterize the biochemical properties of SmaPI purified from extracellular medium.
Main Methods:
- Utilized recombinant Serratia marcescens (pSP2) to study SmaPI production at different temperatures.
- Purified SmaPI from culture supernatant and characterized its pI, molecular mass, stability, and protease specificity.
- Analyzed SmaPI precursor processing, including signal peptide cleavage.
Main Results:
- Supraoptimal temperature (37°C) significantly increased extracellular SmaPI production (86 U/ml) compared to optimal temperature (20 U/ml).
- Purified SmaPI is a monomeric protein (10 kDa) with a pI of ~10.0, stable to boiling, and forms a 1:1 noncovalent complex with SMP.
- SmaPI demonstrated high specificity, inhibiting only SMP among tested proteases.
Conclusions:
- Temperature is a critical factor regulating SmaPI localization and production in Serratia marcescens.
- SmaPI is a thermostable, specific inhibitor of SMP, with potential applications in understanding protease regulation.