title: Development of a denaturation/renaturation-based production process for ferritin nanoparticles.
abstract: Recently, we presented a simple method for generating biological functional protein-based nanoparticles that are ready for use as label agents in bioaffinity assays (Jääskeläinen et al., 2007 Small 3:1362-1367). In this process, the particle shell (ferritin protein) and binding molecules are conjugated via genetic fusion, and particles with binding capacity are produced in a single bacterial cultivation. Production is combined with simple, non-chromatographic purification during which Europium ions are introduced into particles to serve as marker agents. Denaturation-refolding has previously performed by means of pH changes. Here, we test urea as an alternative agent for denaturation, and examine techniques to improve refolding of the functional particles. Three different types of binding molecules were employed in our experiments: biotin carboxyl carrier protein (a small protein with 87 amino acids), single chain antibody fragment (a complex binding protein) and calmodulin-binding peptide (27 amino acids). Urea was successfully utilized to generate functional particles with inherent binding activity and label function. Additionally, particle yield was effectively optimized by analyzing various refolding and bacterial production conditions. Our results clearly demonstrate that this simple biological method of producing functional ferritin-based particles is flexible, and different types of binding moieties can be applied by adjusting the production conditions.
authors.affiliation: Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland.
authors.forename: Anu
authors.initials: A
authors.lastname: Jääskeläinen
authors.forename: Tero
authors.initials: T
authors.lastname: Soukka
authors.forename: Urpo
authors.initials: U
authors.lastname: Lamminmäki
authors.forename: Teemu
authors.initials: T
authors.lastname: Korpimäki
authors.forename: Marko
authors.initials: M
authors.lastname: Virta
mesh_terms: D001709:Biotechnology / Q000379:methods*; D005293:Ferritins / Q000378:metabolism*; D053758:Nanoparticles; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation; D014508:Urea / Q000378:metabolism
chemical_list: D014508:Urea; D005293:Ferritins