High-Throughput Screening of Streptavidin-Binding Proteins in Self-Assembled Solid Films for Directed Evolution of Materials.

Evolution has shaped the development of proteins with an incredible diversity of properties. Incorporating proteins into materials is desirable for applications including biosensing; however, high-throughput selection techniques for screening protein libraries in materials contexts is lacking. In this work, a high-throughput platform to assess the binding affinity for ordered sensing proteins was established. A library of fusion proteins, consisting of an elastin-like polypeptide block, one of 22 variants of rcSso7d, and a coiled-coil order-directing sequence, was generated. All selected variants had high binding in films, likely due to the similarity of the assay to magnetic bead sorting used for initial selection, while solution binding was more variable. From these results, both the assembly of the fusion proteins in their operating state and the functionality of the binding protein are key factors in the biosensing performance. Thus, the integration of directed evolution with assembled systems is necessary to the design of better materials.

Predicting Protein-Polymer Block Copolymer Self-Assembly from Protein Properties.

Protein-polymer bioconjugate self-assembly has attracted a great deal of attention as a method to fabricate protein nanomaterials in solution and the solid state. To identify protein properties that affect phase behavior in protein-polymer block copolymers, a library of 15 unique protein-b-poly(N-isopropylacrylamide) (PNIPAM) copolymers comprising 11 different proteins was compiled and analyzed. Many attributes of phase behavior are found to be similar among all studied bioconjugates regardless of protein properties, such as formation of micellar phases at high temperature and low concentration, lamellar ordering with increasing temperature, and disordering at high concentration, but several key protein-dependent trends are also observed. In particular, hexagonal phases are only observed for proteins within the molar mass range 20-36 kDa, where ordering quality is also significantly enhanced. While ordering is generally found to improve with increasing molecular weight outside of this range, most large bioconjugates exhibited weaker than predicted assembly, which is attributed to chain entanglement with increasing polymer molecular weight. Additionally, order-disorder transition boundaries are found to be largely uncorrelated to protein size and quality of ordering. However, the primary finding is that bioconjugate ordering can be accurately predicted using only protein molecular weight and percentage of residues contained within ß sheets. This model provides a basis for designing protein-PNIPAM bioconjugates that exhibit well-defined self-assembly and a modeling framework that can generalize to other bioconjugate chemistries.

Improved Ordering in Low Molecular Weight Protein-Polymer Conjugates Through Oligomerization of the Protein Block.

The self-assembly of protein-polymer conjugates incorporating oligomers of a small, engineered high-affinity binding protein, rcSso7d.SA, is studied to determine the effect of protein oligomerization on nanoscale ordering. Oligomerization enables a systematic increase in the protein molar mass without changing its overall folded structure, leading to a higher driving force for self-assembly into well-ordered structures. Though conjugates of monomeric rcSso7d.SA are found to only exist in disordered states, oligomers of this protein linked to a poly( N-isopropylacrylamide) (PNIPAM) block self-assemble into lamellar nanostructures. Conjugates of trimeric and tetrameric rcSso7d.SA are observed to produce the strongest ordering in concentrated solution, displaying birefringent lamellae at concentrations as low as 40 wt %. In highly concentrated solution, the oligomeric rcSso7d.SA-PNIPAM block copolymers exhibit ordering and domain spacing trends atypical from that of most block copolymers. Fluorescent binding assays indicate that oligomerized protein blocks retain binding functionality and exhibit limits of detection up to three times lower than that of surface-immobilized protein sensors. Therefore, oligomerization of the protein block in these block copolymers serves as an effective method to improve both nanoscale ordering and biosensing capabilities.

Protein-Polymer Block Copolymer Thin Films for Highly Sensitive Detection of Small Proteins in Biological Fluids.

In nearly all biosensors, sensitivity is greatly reduced for measurements conducted in biological matrices due to nonspecific binding from off-target molecules. One method to overcome this issue is to design a sensor that enables selective size-based uptake of proteins. Herein, a protein-polymer conjugate thin-film biosensor is fabricated that self-assembles into lamellae containing alternating domains of protein and polymer. Analyte is captured in protein regions while polymer domains restrict diffusion of large molecules. Device sensitivity and size-based exclusion properties are probed using two analytes: streptavidin (SA, 52.8 kDa) and monomeric streptavidin (mSA2, 15.6 kDa). Tuning domain spacing by adjusting polymer molecular weight allows the design of films that relatively freely uptake mSA2 and largely restrict SA diffusion. Furthermore, when detecting the smaller mSA2, no reduction in the limit of detection (LOD) is observed when transitioning from detection in the buffer to detection in biological fluids. As a result, LOD measured in fluid samples is reduced by 2 orders of magnitude compared to a traditional surface-immobilized protein monolayer.