title: Low-Tech, Pilot Scale Purification of a Recombinant Spider Silk Protein Analog from Tobacco Leaves.
abstract: Spider dragline is used by many members of the Araneae family not only as a proteinogenic safety thread but also for web construction. Spider dragline has been shown to possess high tensile strength in combination with elastic behavior. This high tensile strength can be attributed to the presence of antiparallel β-sheets within the thread; these antiparallel β-sheets are why the protein is classified as a silk. Due to the properties of spider silk and its technical and medical uses, including its use as a suture material and as a scaffold for tissue regeneration, spider dragline is a focus of the biotechnology industry. The production of sufficient amounts of spider silk is challenging, as it is difficult to produce large quantities of fibers because of the cannibalistic behavior of spiders and their large spatial requirements. In recent years, the heterologous expression of genes coding for spider silk analogs in various hosts, including plants such asNicotiana tabacum, has been established. We developed a simple and scalable method for the purification of a recombinant spider silk protein elastin-like peptide fusion protein (Q-/K-MaSp1-100× ELP) after heterologous production in tobacco leaves involving heat and acetone precipitation. Further purification was performed using centrifugal Inverse Transition Cycling (cITC). Up to 400 mg of highly pure spider silk protein derivatives can be isolated from six kilograms of tobacco leaves, which is the highest amount of silk protein derivatives purified from plants thus far.
authors.affiliation: Department of Downstream Processing, Institute of Pharmacy, Faculty of Sciences I-Biosciences, Martin Luther University Halle-Wittenberg, Weinbergweg 22, Halle 06120, Germany. rene.heppner@pharmazie.uni-halle.de.
authors.forename: René
authors.initials: R
authors.lastname: Heppner
authors.affiliation: Institute of Plant Genetics and Crop Plant Research-IPK, Corrensstrasse 3, Seeland OT Gatersleben 06466, Germany. weichert@ipk-gatersleben.de.
authors.forename: Nicola
authors.initials: N
authors.lastname: Weichert
authors.affiliation: Institute of Biochemistry and Biotechnology, Faculty of Sciences I-Biosciences, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3, Halle 06120, Germany. angelika.schierhorn@biochemtech.uni-halle.de.
authors.forename: Angelika
authors.initials: A
authors.lastname: Schierhorn
authors.affiliation: Institute of Plant Genetics and Crop Plant Research-IPK, Corrensstrasse 3, Seeland OT Gatersleben 06466, Germany. conradu@ipk-gatersleben.de.
authors.forename: Udo
authors.initials: U
authors.lastname: Conrad
authors.affiliation: Department of Downstream Processing, Institute of Pharmacy, Faculty of Sciences I-Biosciences, Martin Luther University Halle-Wittenberg, Weinbergweg 22, Halle 06120, Germany. markus.pietzsch@pharmazie.uni-halle.de.
authors.forename: Markus
authors.initials: M
authors.lastname: Pietzsch
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005349:Fibroins / Q000235:genetics / Q000378:metabolism; D018515:Plant Leaves / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D047011:Silk / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D013112:Spiders / Q000378:metabolism*; D014026:Nicotiana / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D047011:Silk; C086087:spidroin 1; D005349:Fibroins
keywords: downstream processing; pilot scale; purification; scale up; spider silk protein variants