title: The Center for Optimized Structural Studies (COSS) platform for automation in cloning, expression, and purification of single proteins and protein-protein complexes.
abstract: Expression in Escherichia coli represents the simplest and most cost effective means for the production of recombinant proteins. This is a routine task in structural biology and biochemistry where milligrams of the target protein are required in high purity and monodispersity. To achieve these criteria, the user often needs to screen several constructs in different expression and purification conditions in parallel. We describe a pipeline, implemented in the Center for Optimized Structural Studies, that enables the systematic screening of expression and purification conditions for recombinant proteins and relies on a series of logical decisions. We first use bioinformatics tools to design a series of protein fragments, which we clone in parallel, and subsequently screen in small scale for optimal expression and purification conditions. Based on a scoring system that assesses soluble expression, we then select the top ranking targets for large-scale purification. In the establishment of our pipeline, emphasis was put on streamlining the processes such that it can be easily but not necessarily automatized. In a typical run of about 2 weeks, we are able to prepare and perform small-scale expression screens for 20-100 different constructs followed by large-scale purification of at least 4-6 proteins. The major advantage of our approach is its flexibility, which allows for easy adoption, either partially or entirely, by any average hypothesis driven laboratory in a manual or robot-assisted manner.
authors.affiliation: Department of Structural and Computational Biology, Max F. Perutz Laboratories, University of Vienna, Campus Vienna Biocenter 5, 1030, Vienna, Austria.
authors.forename: Georg
authors.initials: G
authors.lastname: Mlynek
authors.forename: Anita
authors.initials: A
authors.lastname: Lehner
authors.forename: Jana
authors.initials: J
authors.lastname: Neuhold
authors.forename: Sarah
authors.initials: S
authors.lastname: Leeb
authors.forename: Julius
authors.initials: J
authors.lastname: Kostan
authors.forename: Alexej
authors.initials: A
authors.lastname: Charnagalov
authors.forename: Peggy
authors.initials: P
authors.lastname: Stolt-Bergner
authors.forename: Kristina
authors.initials: K
authors.lastname: Djinović-Carugo
authors.forename: Nikos
authors.initials: N
authors.lastname: Pinotsis
mesh_terms: D057205:Automation, Laboratory; D002850:Chromatography, Gel / Q000379:methods; D003001:Cloning, Molecular; D019976:Cloning, Organism; D019295:Computational Biology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D064448:Filamins / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification*
chemical_list: D064448:Filamins; D011994:Recombinant Proteins