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Abstract Affinity precipitation is a powerful separation method in that it combines the binding selectivity of affinity chromatography with precipitation of captured biomolecules via phase separation triggered by small changes in the environment, e.g., pH, ionic strength, temperature, light, etc. Elastin‐like polypeptides (ELPs) are thermally responsive biopolymers composed of pentapeptide repeats VPGVG that undergo reversible phase separation, where they aggregate when temperature and/or salt concentration are increased. Here we describe the generation of an ELP fusion to a soluble streptavidin mutant that enables rapid purification of anyStrep‐tag II fusion protein of interest. This heterobifunctional protein takes advantage of the native tetrameric structure of streptavidin, leading to binding‐induced multivalent crosslinking upon protein capture. The efficient biotin‐mediated dissociation of the boundStrep‐tag II fusion protein from the streptavidin‐ELP capturing scaffold allows for mild elution conditions. We also show that this platform is particularly effective in the purification of a virus‐like particle (VLP)‐like E2 protein nanoparticle, likely because the high valency of the protein particle causes binding‐induced crosslinking and precipitation. Considering the importance of VLP for gene therapy applications, we believe this is a particularly exciting advance. We demonstrated this feasibility by the efficient purification of a VLP‐like E2 protein nanoparticle as a surrogate.
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Stable, small, specific, low-valency quantum dots for single-molecule imaging
We have developed a strategy for synthesizing immediately activable, water-soluble, compact (∼10–12 nm hydrodynamic diameter) quantum dots with a small number of stable and controllable conjugation handles for long distance delivery and subsequent biomolecule conjugation. Upon covalent conjugation with engineered monovalent streptavidin, the sample results in a population consisting of low-valency quantum dots. Alternatively, we have synthesized quantum dots with a small number of biotin molecules that can self-assemble with engineered divalent streptavidin via high-affinity biotin–streptavidin interactions. Being compact, stable and highly specific against biotinylated proteins of interest, these low-valency quantum dots are ideal for labeling and tracking single molecules on the cell surface with high spatiotemporal resolution for different biological systems and applications.
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- Award ID(s):
- 1653782
- PAR ID:
- 10082949
- Date Published:
- Journal Name:
- Nanoscale
- Volume:
- 10
- Issue:
- 9
- ISSN:
- 2040-3364
- Page Range / eLocation ID:
- 4406 to 4414
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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null (Ed.)As potential high surface area for selective capture in diagnostic or filtration devices, biotin-cellulose nanofiber membranes were fabricated to demonstrate the potential for specific and bio-orthogonal attachment of biomolecules onto nanofiber surfaces. Cellulose acetate was electrospun and substituted with alkyne groups in either a one- or two-step process. The alkyne reaction, confirmed by FTIR and Raman spectroscopy, was dependent on solvent ratio, time, and temperature. The two-step process maximized alkyne substitution in 10/90 volume per volume ratio (v/v) water to isopropanol at 50 °C after 6 h compared to the one-step process in 80/20 (v/v) at 50 °C after 48 h. Azide-biotin conjugate “clicked” with the alkyne-cellulose via copper-catalyzed alkyne-azide cycloaddition (CuAAC). The biotin-cellulose membranes, characterized by FTIR, SEM, Energy Dispersive X-ray spectroscopy (EDX), and XPS, were used in proof-of-concept assays (HABA (4′-hydroxyazobenzene-2-carboxylic acid) colorimetric assay and fluorescently tagged streptavidin assay) where streptavidin selectively bound to the pendant biotin. The click reaction was specific to alkyne-azide coupling and dependent on pH, ratio of ascorbic acid to copper sulfate, and time. Copper (II) reduction to copper (I) was successful without ascorbic acid, increasing the viability of the click conjugation with biomolecules. The surface-available biotin was dependent on storage medium and time: Decreasing with immersion in water and increasing with storage in air.more » « less
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We demonstrate a higher sensitivity detection of proteins in a photonic crystal platform by including a deep subwavelength feature in the unit cell that locally increases the energy density of light. Through both simulations and experiments, the sensing capability of a deep subwavelength-engineered silicon antislot photonic crystal nanobeam (PhCNB) cavity is compared to that of a traditional PhCNB cavity. The redistribution and local enhancement of the energy density by the 50 nm antislot enable stronger light–molecule interaction at the surface of the antislot and lead to a larger resonance shift upon protein binding. This surface-based energy enhancement is confirmed by experiments demonstrating a nearly 50% larger resonance shift upon attachment of streptavidin molecules to biotin-functionalized antislot PhCNB cavities.more » « less
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/C7NR08673C
