continuing emphasis. Polypropylene (PP) capillary-channeled polymer (C-CP) fiber columns are modified with the biotin- binding protein streptavidin (SAV) to capture biotinylated proteins. The loading characteristics of SAV on fiber supports were determined using breakthrough curves and frontal analysis. Based on adsorption data, a 3-min on-column loading at a flow rate of 0.5 mL min−1 (295.2 cm h−1) with a SAV feed concentration of 0.5 mg mL−1 produces a SAV loading capacity of 1.4 mg g−1 fiber. SAV has an incredibly high affinity for the small-molecule biotin (10−14 M), such that this binding relationship can be exploited by labeling a target protein with biotin via an Avi-tag. To evaluate the capture of the biotinylated proteins on the modified PP surface, the biotinylated versions of bovine serum albumin (b-BSA) and green fluorescent protein (b-GFP) were utilized as probe species. The loading buffer composition and flow rate were optimized towards protein capture. The non-ionic detergent Tween-20 was added to the deposition solutions to minimize non-specific binding. Values of 0.25–0.50% (v/v) Tween-20 in PBS exhibited better capture efficiency, while minimizing the non-specific binding for b-BSA and b-GFP, respectively. The C-CP fiber platform has the potential to provide a fast and low-cost method to capture targeted proteins for applications including protein purification or pull-down assays.
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This content will become publicly available on January 10, 2025
Ligand-Mediated Mechanical Enhancement in Protein Complexes at Nano- and Macro-Scale
Protein self-assembly plays a vital role in a myriad of biological functions and in the construction of biomaterials. Although the physical association underlying these assemblies offers high specificity, the advantage often compromises the overall durability of protein complexes. To address this challenge, we propose a novel strategy that reinforces the molecular self-assembly of protein complexes mediated by their ligand. Known for their robust noncovalent interactions with biotin, streptavidin (SAv) tetramers are examined to understand how the ligand influences the mechanical strength of protein complexes at the nanoscale and macroscale, employing atomic force microscopy-based single-molecule force spectroscopy, rheology, and bioerosion analysis. Our study reveals that biotin binding enhances the mechanical strength of individual SAv tetramers at the nanoscale. This enhancement translates into improved shear elasticity and reduced bioerosion rates when SAv tetramers are utilized as cross-linking junctions within hydrogel. This approach, which enhances the mechanical strength of protein-based materials without compromising specificity, is expected to open new avenues for advanced biotechnological applications, including self-assembled, robust biomimetic scaffolds and soft robotics.
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- NSF-PAR ID:
- 10501131
- Publisher / Repository:
- American Chemical Society
- Date Published:
- Journal Name:
- ACS Applied Materials & Interfaces
- Volume:
- 16
- Issue:
- 1
- ISSN:
- 1944-8244
- Page Range / eLocation ID:
- 272 to 280
- Subject(s) / Keyword(s):
- protein complex ligand artificial protein design molecular self-assembly nanomechanics hydrogel bulk mechanical properties
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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