Expression and Simple Purification Strategy for the Generation of Anti-microbial Active Enterocin P from Enterococcus faecium Expressed in Escherichia coli ER2566

Abstract

Background: Enterocin-P of Enterococcus faecium P13 (EntP) is of great interest as a food preservative and medicine due to its non-toxicity and broad antimicrobial spectrum in various pH, as well as its excellent thermal stability. However, recombinant production of EntP is still in laboratory because of low productivity and complex purification process. Objectives: In this study, we aimed to develop efficient methods for high-level expression and convenient purification of the recombinant EntP. Materials and Methods: An artificially synthesized gene (entP) of 132 bp encoding mature enterocin P of E. faecium P13 was cloned in plasmid pTWIN1 under the control of an inducible T7lac promoter for expression of fusion protein EntP-Mxe GyrA mini-intein-chitin binding domain (CBD) (abbreviated by EntP-Int-CBD) in E. coli. Recombinant EntP was released from the fusion protein by DTT digestion and cleaned by distill water and checked for anti-bacterial activity. Results: The fusion protein was highly expressed in insoluble form in E. coli at 37oC with 0.05 mM IPTG induction. The insoluble fusion protein EntP-Int-CBD was easily prepared by cell sonication and centrifugation to remove soluble contaminants. The repeat washing steps with Triton X-100 were applied to reduce contaminants. After DTT-induced self-digestion in urea 4 M, the EntP released from the fusion protein was insoluble in water and easier to be separated from soluble Int-CBD by centrifugation. The recombinant peptide was soluble in 20% 2-propanol in 0.1% trifluoroacetic acid (TFA) and exhibited strong anti- Listeria monocytogenes and Staphylococcus aureus activities. Conclusion: This study is the first report providing a simple, quick and straight forward procedure for heterologous production of functional and pure Enterocin P without using any chromatography columns in the purification process.