Ducka Paulina, Eckhard Ulrich, Schönauer Esther, Kofler Stefan, Gottschalk Gerhard, Brandstetter Hans, Nüss Dorota
Department of Molecular Biology, University of Salzburg, Austria.
Appl Microbiol Biotechnol. 2009 Jul;83(6):1055-65. doi: 10.1007/s00253-009-1953-4. Epub 2009 Mar 31.
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.
一方面，这些酶能使致病性梭菌实现宿主浸润和定殖；另一方面，由于它们具有降解各种类型胶原蛋白和明胶的能力，因而成为了有价值的生物技术工具。然而，由于其致病源以及纯合同工酶的复杂纯化过程，对高质量制剂的需求超过了供应。我们展示了一种大肠杆菌表达系统的建立，该系统用于表达来自溶组织梭菌的多种胶原酶G（ColG）和H（ColH）构建体以及来自破伤风梭菌的胶原酶T（ColT）构建体，模拟体内同工酶。基于五种不同表达菌株和两种表达载体的设置，表达了12种不同的构建体，并建立了一个灵活的纯化平台，该平台由各种适用于各个变体个体需求的正交色谱步骤组成。这个快速、经济高效且易于建立的平台使我们能够每升培养物获得至少10毫克的高纯度单同工型蛋白，非常适合众多复杂的下游应用。这个生产和纯化平台为重组胶原酶的系统筛选铺平了道路，以阐明其生化功能并确定胶原降解中的关键残基和基序。
