title: Flow-cytometric screening of aggregation-inhibitors using a fluorescence-assisted intracellular method.
abstract: Aggregation of misfolded peptides and proteins is a key event in several neurodegenerative diseases. Suggested treatments of such disorders aim to inhibit the initial aggregation process. Here, we have developed an intracellular, function-based screening method, intended for isolation of aggregation-inhibitors from combinatorial protein libraries by flow-cytometric cell sorting. The method is based on fusion of aggregation-prone peptides to a fluorescent protein, functioning as a solubility reporter. Co-expression of a protein-based aggregation-inhibitor should prevent aggregation and thus increase the whole-cell fluorescence. We evaluated the method using the aggregation-prone Alzheimer's-related amyloid-β (Aβ) peptide in fusion to green-fluorescent protein (GFP), and an Aβ aggregation-inhibiting Affibody molecule. To adapt the method for library applications, the inhibitor was linked to an mCherry reporter for normalization of protein expression levels. We found that aggregation propensity correlates with fluorescence intensity, as co-expression of the Affibody-inhibitor increased the whole-cell fluorescence relative to a non-inhibitor. Employing improved cultivation parameters, we furthermore demonstrated efficient rescue from aggregation of an α-synuclein-derived protein using a different type of aggregation-inhibitor. Importantly, we also showed that the Aβ aggregation-inhibiting Affibody molecule could be isolated from a 1:10,000 background of non-inhibitors, with around 3,500-fold enrichment, in one cycle of fluorescence-based cell sorting. In conclusion, our new method represents a promising approach for generation of novel protein-based aggregation-inhibitors.
authors.affiliation: Division of Protein Technology, School of Biotechnology, KTH - Royal Institute of Technology, Stockholm, Sweden.
authors.forename: Hanna
authors.initials: H
authors.lastname: Lindberg
authors.affiliation: Division of Protein Technology, School of Biotechnology, KTH - Royal Institute of Technology, Stockholm, Sweden.
authors.forename: Lisa
authors.initials: L
authors.lastname: Sandersjöö
authors.affiliation: Division of Protein Technology, School of Biotechnology, KTH - Royal Institute of Technology, Stockholm, Sweden.
authors.forename: Sebastian W
authors.initials: SW
authors.lastname: Meister
authors.affiliation: Division of Proteomics and Nanobiotechnology, Science for Life Laboratory, School of Biotechnology, KTH - Royal Institute of Technology, Solna, Sweden.
authors.forename: Mathias
authors.initials: M
authors.lastname: Uhlén
authors.affiliation: Division of Protein Technology, School of Biotechnology, KTH - Royal Institute of Technology, Stockholm, Sweden.
authors.forename: John
authors.initials: J
authors.lastname: Löfblom
authors.affiliation: Division of Protein Technology, School of Biotechnology, KTH - Royal Institute of Technology, Stockholm, Sweden.
authors.forename: Stefan
authors.initials: S
authors.lastname: Ståhl
mesh_terms: D016229:Amyloid beta-Peptides / Q000737:chemistry / Q000378:metabolism; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D005434:Flow Cytometry / Q000379:methods*; D005453:Fluorescence; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D051844:alpha-Synuclein / Q000737:chemistry / Q000378:metabolism
chemical_list: D016229:Amyloid beta-Peptides; D011994:Recombinant Proteins; D051844:alpha-Synuclein; D049452:Green Fluorescent Proteins
keywords: Affibody molecules; Aggregation-inhibitor; Amyloid beta; Combinatorial protein engineering; Intracellular selection system