Construction and Validation of a New Naïve Sequestrin Library for Directed Evolution of Binders against Aggregation-Prone Peptides

Abstract

Affibody molecules are small affinity proteins that have excellent properties for many different applications, ranging from biotechnology to diagnostics and therapy. The relatively flat binding surface is typically resulting in high affinity and specificity when developing binding reagents for globular target proteins. For smaller unstructured peptides, the paratope of affibody molecules makes it more challenging to achieve a sufficiently large binding surface for high-affinity interactions. Here, we describe the development of a new type of protein scaffold based on a dimeric form of affibodies with a secondary structure content and mode of binding that is distinct from conventional affibody molecules. The interaction is characterized by encapsulation of the target peptide in a tunnel-like cavity upon binding. The new scaffold was used for construction of a high-complexity phage-displayed library and selections from the library against the amyloid beta peptide resulted in identification of high-affinity binders that effectively inhibited amyloid aggregation.

Keywords: Alzheimer’s disease; Aβ; affibody; directed evolution; phage display; sequestrins.

Figure 1

(A) Structure of conventional affibody molecule with randomized positions marked in beige for comparison (PDB:2B89). (B) Two subunits of Z_Aβ3 [6] (light purple and blue) in complex with amyloid beta (Aβ) (grey) (PDB:2OTK). A cysteine bridge between the two subunits is shown as sticks. (C) The head-to-tail Sq_lib with the 22 randomized positions shown in beige for the two subunits. (D) The randomised positions in the design and with connecting linker. Each position group has a specified distribution of amino acids. The same distribution is used for both subunits.

Figure 2

Library distribution in percentage for Sq_lib in the (A) first subunit and (B) second subunit at randomized positions. Generally, the design is maintained in the created library pool.

Figure 3

Sodium Dodecyl Sulphate–Polyacrylamide Gel (SDS-PAGE) analysis of produced sequestrins Sq_Aβ with 1.5 μg loaded per construct, at expected weight of 13 kDa. (A) Sequestrins from third and fourth cycle. LMW: low molecular weight ladder. (B) Sequestrins from fifth cycle. * Lanes intentionally left empty.

Figure 4

Surface plasmon resonance (SPR) sensorgram for the sequestrins Sq_Aβ22, and Sq_Aβ23 at (A,B) 25 °C, and (C,D) 37 °C, respectively, and their corresponding kinetic constants. The y-axis shows the relative response units (RU) and x-axis time(s). Kinetics were recorded for 900 s, with analyte injection ending at 200 s. The sequestrins were injected in duplicate in a dilution series spanning 1:1.5 steps from 342 to 30 nM for the 25 °C affinity measurement. At 37 °C the sequestrins were injected in triplicates with concentrations 300–75 nM in a 1:2 dilution series. The black lines represent the fitting of the data to calculate kinetics. The experiment was conducted with immobilized Aβ_1–40 at a coating density of 80 RU.

Figure 5

Circular dichroism (CD) spectra between 195–260 nm for proteins in equimolar concentrations of 15.7 μM for (A,B) Indicates the structural rearrangement upon co-incubation of peptide and sequestrins (light purple). The green line shows the signal lost upon co-incubation between Aβ (beige) and the sequestrin (blue). (C,D) Variable temperature measurements at 221 nm of Sq_Aβ22 and Sq_Aβ23 with and without Aβ co-incubated with the sequestrins. (E,F) Sq_Aβ22 and Sq_Aβ23 in complex with Aβ before (light purple) and after (blue) thermal melting.

Figure 6

Aggregation assay measuring thioflavin T (ThT) fluorescence at 480 nm. (A) Histogram showing endpoint ThT fluorescence at 92 h for 20 μM Aβ_1–40, 20 μM Sq_Aβ22, 20 μM Sq_Aβ23, 1:1 molar ratio of 20 μM Aβ_1–40 + 20 μM Sq_Aβ22 and 1:1 molar ratio of 20 μM Aβ_1–40 + 20 μM Sq_Aβ23. (B) Histogram showing endpoint ThT fluorescence at 92 h for 20 μM Aβ_1–42, 20 μM Sq_Aβ22, 20 μM Sq_Aβ23, 1:1 20 μM Aβ_1–42 + 20 μM Sq_Aβ22 and 1:1 20 μM Aβ_1–42 + 20 μM Sq_Aβ23. (C) ThT fluorescence monitored for 92 h for 20 μM Aβ_1–42 (grey), 1:1 molar ratio of 20 μM Aβ_1–40 + 20 μM Sq_Aβ22 (blue) and 1:1 molar ratio of 20 μM Aβ_1–40 + 20 μM Sq_Aβ23 (purple). (D) Histogram showing endpoint ThT fluorescence at 92 h for 12–20 μM Aβ_1–40 (in grey), and sequestrins Sq_Aβ22 (blue) and Sq_Aβ23 (purple) at different molar ratios of 1:1, 1:5 and 1:10 in 20 μM Aβ_1–42. Standard deviation of the replicates is shown in the graph.