Flow‐cytometric screening of aggregation‐inhibitors using a fluorescence‐assisted intracellular method

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. Aggregation of misfolded peptides or proteins is believed to be the primary event in several neurodegenerative disorders, and inhibition of the initial aggregation process is an attractive strategy for treatment. The development of a novel method for selection of protein‐based aggregation‐inhibitors is reported. The method is based on fusion of the aggregation‐prone target to green fluorescent protein, functioning as a solubility reporter, and fusion of the inhibitor to an mCherry reporter for normalization of the protein expression levels. By optimization of cultivation parameters the efficient FACS‐mediated isolation an Aβ aggregation‐inhibiting Affibody molecule from a 1:10 000 background of non‐inhibitors with a 3500‐fold enrichment in one single round of sorting is shown.