Cloning, expression and purification of extracellular serine protease Esp, a biofilm‐degrading enzyme, from Staphylococcus epidermidis

• Published in: Journal of applied microbiology Vol. 111; no. 6; pp. 1406 - 1415
• Main Authors: Sugimoto, S, Iwase, T, Sato, F, Tajima, A, Shinji, H, Mizunoe, Y
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.12.2011; Blackwell
• Subjects: affinity chromatography; biofilm; biofilm(s); Biofilms; Biological and medical sciences; biotechnology; Brevibacillus; Brevibacillus - genetics; Brevibacillus - metabolism; Brevibacillus choshinensis; Caseins - metabolism; Chromatography, Affinity; Cloning, Molecular; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Fundamental and applied biological sciences. Psychology; genes; Histidine - chemistry; Microbiology; nickel; nose; Plasmids; Protein Sorting Signals; proteinase; proteolysis; purification methods; rapid methods; recombinant proteins; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Serine Proteases - chemistry; Serine Proteases - genetics; Serine Proteases - isolation & purification; serine proteinases; signal peptide; staphylococci; Staphylococcus aureus; Staphylococcus aureus - physiology; Staphylococcus epidermidis; Staphylococcus epidermidis - enzymology; Staphylococcus epidermidis - genetics; structural proteins; Thermolysin - metabolism; Biotechnology; Purification; Serine endopeptidases; Enzyme; Applied microbiology; rapid methods; staphylococci; Method; Extracellular; biofilm(s); Degradation; Peptidases; Staphylococcus epidermidis; proteinase; Biofilm; Bacteria; Hydrolases; Micrococcales; Micrococcaceae
• ISSN: 1364-5072; 1365-2672
• DOI: 10.1111/j.1365-2672.2011.05167.x

Aims: Staphylococcus epidermidis Esp, an extracellular serine protease, inhibits Staphylococcus aureus biofilm formation and nasal colonization. To further expand the biotechnological applications of Esp, we developed a highly efficient and economic method for the purification of recombinant Esp based on a Brevibacillus choshinensis expression–secretion system. Methods and Results: The esp gene was fused with the N‐terminal Sec‐dependent signal sequence of the B. choshinensis cell wall protein and a C‐terminal hexa‐histidine‐tag gene. The recombinant Esp was expressed and secreted into the optimized medium as an immature form and subsequently activated by thermolysin. The mature Esp was easily purified by a single purification step using nickel affinity chromatography and showed proteolytic activity as well as Staph. aureus biofilm destruction activity. Conclusions: The purification yield of the developed extracellular production system was 5 mg recombinant mature Esp per 20‐ml culture, which was much higher than that of an intracellular production system in Escherichia coli (3 mg recombinant Esp per 1‐l culture). Significance and Impact of the Study: Our findings will be a powerful tool for the production and purification of recombinant Esp and also applicable to a large variety of recombinant proteins used for basic researches and biotechnological applications.