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The Ebola virus is a deadly pathogen that has been threatening public health for decades. Recent studies have revealed alternative viral invasion routes where Ebola virus approaches cells via interactions among phosphatidylserine (PS), PS binding ligands such as Gas6, and TAM family receptors such as Axl. In this study, we investigate the interactions among phosphatidylserine on the Ebola viral-like particle (VLP) membrane, human Gas6, and human Axl using atomic force microscope-based single molecule force spectroscopy to compare their binding strength and affinity from a biomechanical perspective. The impact of calcium ions on their interactions is also studied and quantified to provide more details on the calcium-dependent phosphatidylserine-Gas6 binding mechanism. Our results indicate that, in the presence of calcium ions, the binding strengths of VLP-Gas6 and VLP-Gas6-Axl increase but are still weaker than that of Gas6-Axl, and the binding affinity of VLP-Gas6 and VLP-Gas6-Axl is largely improved. The binding strength and affinity of Gas6-Axl basically remain the same, indicating no impact in the presence of calcium ions. Together, our study suggests that, under physiological conditions with calcium present, the Ebola virus can utilize its membrane phosphatidylserine to dock on cell surface via Gas6-Axl bound complex.
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This content will become publicly available on December 1, 2025
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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