title: Heterologous expression and pro-peptide supported refolding of the high specific endopeptidase Lys-C.
abstract: The high specific lysyl endopeptidase (Lys-C; EC 3.4.21.50) is often used for the initial fragmentation of polypeptide chains during protein sequence analysis. However, due to its specificity it could be a useful tool for the production of tailor-made protein hydrolysates with for example bioactive or techno functional properties. Up to now, the high price makes this application nearly impossible. In this work, the increased expression for Escherichia coli optimized Lys-C was investigated. The cloned sequence had a short artificial N-terminal pro-peptide (MGSK). The expression of MGSK-Lys-C was tested using three expression vectors and five E. coli host strains. The highest expression rate was obtained for the expression system consisting of the host strain E. coli JM109 and the rhamnose inducible expression vector pJOE. A Lys-C activity of 9340 ± 555 nkatTos-GPK-pNA/Lculture could be achieved under optimized cultivation conditions after chemical refolding. Furthermore, the influence of the native pre-N-pro peptide of Lys-C from Lysobacter enzymogenes ssp. enzymogenes ATCC 27796 on Lys-C refolding was investigated. The pre-N-pro peptide was expressed recombinantly in E. coli JM109 using the pJOE expression vector. The optimal concentration of the pre-N-pro peptide in the refolding procedure was 100 μg/mLrefolding buffer and the Lys-C activity could be increased to 541,720 nkatTos-GPK-pNA/Lculture. With the results presented, the expensive lysyl endopeptidase can be produced in high activity and high amounts and the potential of Lys-C for tailor-made protein hydrolysates with bioactive (e.g. antihypertensive) and/or techno functional (e.g. foaming, emulsifying) properties can be investigated in future time studies.
authors.affiliation: Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Stuttgart, Germany. Electronic address: t.stressler@uni-hohenheim.de.
authors.forename: Timo
authors.initials: T
authors.lastname: Stressler
authors.affiliation: Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Stuttgart, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Eisele
authors.affiliation: Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Stuttgart, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Meyer
authors.affiliation: Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Stuttgart, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Wangler
authors.affiliation: Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Stuttgart, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Hug
authors.affiliation: Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Stuttgart, Germany.
authors.forename: Sabine
authors.initials: S
authors.lastname: Lutz-Wahl
authors.affiliation: Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Stuttgart, Germany.
authors.forename: Lutz
authors.initials: L
authors.lastname: Fischer
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D053528:Lysobacter / Q000201:enzymology*; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D058849:Protein Refolding
chemical_list: D001426:Bacterial Proteins; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase
keywords: Cultivation; Escherichia coli; Lysobacter enzymogenes; Lysyl endopeptidase; Pro-peptide; Refolding