A spidroin‐derived solubility tag enables controlled aggregation of a designed amyloid protein

Abstract

Amyloidogenesis is associated with more than 30 diseases, but the molecular mechanisms involved in cell toxicity and fibril formation remain largely unknown. The inherent tendency of amyloid-forming proteins to aggregate renders expression, purification, and experimental studies challenging. NT* is a solubility tag derived from a spider silk protein that was recently introduced for the production of several aggregation-prone peptides and proteins at high yields. Herein, we investigate whether fusion to NT* can prevent amyloid fibril formation and enable controlled aggregation for experimental studies. As an example of an amyloidogenic protein, we chose the de novo-designed polypeptide β17. The fusion protein NT*-β17 was recombinantly expressed in Escherichia coli to produce high amounts of soluble and mostly monomeric protein. Structural analysis showed that β17 is kept in a largely unstructured conformation in fusion with NT*. After proteolytic release, β17 adopts a β-sheet conformation in a pH- and salt-dependent manner and assembles into amyloid-like fibrils. The ability of NT* to prevent premature aggregation and to enable structural studies of prefibrillar states may facilitate investigation of proteins involved in amyloid diseases.

Augmentation of Bri2 molecular chaperone activity against amyloid-β reduces neurotoxicity in mouse hippocampus in vitro

“…Rh Bri2 BRICHOS R221E was produced in fusion with a solubility tag, NT*, that was recently developed based on the Nterminal domain of spider silk proteins 26,41,42 . Purified NT*-Bri2 BRICHOS R221E was separated into oligomers, dimers, and monomers by size-exclusion chromatography (SEC; Fig.…”

Functionalization of amyloid fibrils via the Bri2 BRICHOS domain

“…For in vitro analysis of binding properties of rh Bri2 BRICHOS, we generated amyloid fibrils of Aβ 42 , IAPP and α-synuclein, which are implied in Alzheimer’s disease, type II diabetes and Parkinson’s disease, respectively, as well as the de novo-designed β17 protein, according to published protocols 18 , 25 , 30 , 31 . The fibrils were isolated by centrifugation and subsequently incubated with rh Bri2 BRICHOS-mCherry at a concentration that corresponds to 20% of the monomeric amyloid polypeptide concentration, at 37 °C for one hour.…”

“…Expression and purification of rh Bri2 BRICHOS-mCherry were done as previously described for rh Bri2 BRICHOS 21 . Aβ42 and β17 were expressed and purified as described previously 25 , 30 . Expression of NT* FlSp (TEV recognition site, TRS)-α-synuclein and NT* FlSp MetIAPP was performed as described previously 25 , 30 , 34 .…”

“…Aβ42 and β17 were expressed and purified as described previously 25 , 30 . Expression of NT* FlSp (TEV recognition site, TRS)-α-synuclein and NT* FlSp MetIAPP was performed as described previously 25 , 30 , 34 . Purification of αSN was performed essentially as described earlier for Aβ 25 .…”

Synthetic surfactants with SP‐B and SP‐C analogues to enable worldwide treatment of neonatal respiratory distress syndrome and other lung diseases

“…One approach to improve the water solubility of hydrophobic proteins is the use of solubility tags during the expression and purification steps, and several different types of solubility tags have been introduced . We have recently developed a novel solubility tag that is efficient compared to other tags tested for the production of SP‐C33Leu, other non‐polar peptides, as well as several proteins that are prone to aggregate for various reasons . This tag is based on the idea that spider silk proteins (spidroins) that are prone to aggregate into silk but stored in solution at very high concentrations, would need a natural solubility tag.…”