title: A universal strategy for high-yield production of soluble and functional clostridial collagenases in E. coli.
abstract: Clostridial collagenases are foe and friend: on the one hand, these enzymes enable host infiltration and colonization by pathogenic clostridia, and on the other hand, they are valuable biotechnological tools due to their capacity to degrade various types of collagen and gelatine. However, the demand for high-grade preparations exceeds supply due to their pathogenic origin and the intricate purification of homogeneous isoforms. We present the establishment of an Escherichia coli expression system for a variety of constructs of collagenase G (ColG) and H (ColH) from Clostridium histolyticum and collagenase T (ColT) from Clostridium tetani, mimicking the isoforms in vivo. Based on a setup of five different expression strains and two expression vectors, 12 different constructs were expressed, and a flexible purification platform was established, consisting of various orthogonal chromatography steps adaptable to the individual needs of the respective variant. This fast, cost-effective, and easy-to-establish platform enabled us to obtain at least 10 mg of highly pure mono-isoformic protein per liter of culture, ideally suited for numerous sophisticated downstream applications. This production and purification platform paves the way for systematic screenings of recombinant collagenases to enlighten the biochemical function and to identify key residues and motifs in collagenolysis.
authors.affiliation: Department of Molecular Biology, University of Salzburg, Austria.
authors.forename: Paulina
authors.initials: P
authors.lastname: Ducka
authors.forename: Ulrich
authors.initials: U
authors.lastname: Eckhard
authors.forename: Esther
authors.initials: E
authors.lastname: Schönauer
authors.forename: Stefan
authors.initials: S
authors.lastname: Kofler
authors.forename: Gerhard
authors.initials: G
authors.lastname: Gottschalk
authors.forename: Hans
authors.initials: H
authors.lastname: Brandstetter
authors.forename: Dorota
authors.initials: D
authors.lastname: Nüss