Flow-cytometric screening of aggregation-inhibitors using a fluorescence-assisted intracellular method
2017 (English)In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 12, no 1, 1600364Article in journal (Refereed) Published
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.
Place, publisher, year, edition, pages
John Wiley & Sons, 2017. Vol. 12, no 1, 1600364
Affibody molecules, Aggregation-inhibitor, Amyloid beta, Combinatorial protein engineering, Intracellular selection system, Cells, Cytology, Diagnosis, Fluorescence, Glycoproteins, Molecules, Neurodegenerative diseases, Peptides, Aggregation inhibitors, Amyloid betas, Protein engineering, Selection systems, Proteins, alpha synuclein, amyloid beta protein, green fluorescent protein, recombinant protein, chemistry, Escherichia coli, flow cytometry, gene vector, genetics, metabolism, preclinical study, procedures, alpha-Synuclein, Amyloid beta-Peptides, Drug Evaluation, Preclinical, Genetic Vectors, Green Fluorescent Proteins, Recombinant Proteins
IdentifiersURN: urn:nbn:se:kth:diva-202248DOI: 10.1002/biot.201600364ScopusID: 2-s2.0-85006293410OAI: oai:DiVA.org:kth-202248DiVA: diva2:1081108
Funding text: We thank Affibody AB for providing the genetic construct of ZHER2-mCherry. The Wallenberg Center for Protein Research and the Swedish Brain Foundation (grant FO2015-0174) are acknowledged for funding. Conceived and designed the experiments: HL LS JL SS. Performed the experiments: HL LS SM. Contributed reagents/materials/analysis tools and analyzed the data: HL LS SM MU JL SS. Wrote the paper: HL LS JL SS. The authors declare no conflicts of interest. QC 201703132017-03-132017-03-132017-03-15Bibliographically approved