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Biofilm-related infections are associated with high mortality and morbidity combined with increased treatment costs. Traditional antibiotics are becoming less effective due to the emergence of drug-resistant bacterial strains. The need to treat biofilms on medical implants is particularly acute, and one persistent challenge is selectively directing nanoparticles to the biofilm site. Here, we present a protein-based functionalization strategy that targets the extracellular matrix of biofilms. The engineered protein combines the Staphylococcus epidermidis autolysin R2ab domain with a gold-binding GB3 domain, directing nanoparticles specifically to bacterial cell wall components (lipoteichoic acid and wall teichoic acid) that are absent in mammalian tissues. This fusion protein is applied to a gold nanoparticle (AuNP) core, along with elastin-like polypeptides (ELPs), which generate a robust photothermal response. The engineered particles exhibit exceptional biocompatibility, including low protein corona formation, minimal macrophage uptake, and hemocompatibility, while maintaining selective biofilm targeting. The photothermal conversion can be modulated by changing the ELP transition temperature, and the functionalized AuNPs strongly interact with biofilms under static and flow conditions without significantly binding to serum-coated surfaces. Near-infrared laser irradiation resulted in a 10,000-fold improvement in killing efficiency compared to untreated controls (p < 0.0001). The targeting strategy utilized here represents a versatile approach to targeting drug-resistant infections and could be readily expanded to other bacterial pathogens and anti-biofilm nanoparticle platforms.
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Engineering of heterobifunctional biopolymers for tunable binding and precipitation of Strep‐Tag proteins and virus‐like nanoparticles
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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- Award ID(s):
- 2040749
- PAR ID:
- 10623897
- Publisher / Repository:
- Wiley
- Date Published:
- Journal Name:
- Biotechnology and Bioengineering
- Volume:
- 121
- Issue:
- 12
- ISSN:
- 0006-3592
- Page Range / eLocation ID:
- 3860 to 3868
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1002/bit.28845
