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SpySwitch enables pH- or heat-responsive capture and release for plug-and-display nanoassembly.

ABSTRACT: Proteins can be empowered via SpyTag for anchoring and nanoassembly, through covalent bonding to SpyCatcher partners. Here we generate a switchable version of SpyCatcher, allowing gentle purification of SpyTagged proteins. We introduce numerous histidines adjacent to SpyTag's binding site, giving moderate pH-dependent release. After phage-based selection, our final SpySwitch allows purification of SpyTag- and SpyTag003-fusions from bacterial or mammalian culture by capture at neutral pH and release at pH 5, with purity far beyond His-tag methods. SpySwitch is also thermosensitive, capturing at 4 °C and releasing at 37 °C. With flexible choice of eluent, SpySwitch-purified proteins can directly assemble onto multimeric scaffolds. 60-mer multimerization enhances immunogenicity and we use SpySwitch to purify receptor-binding domains from SARS-CoV-2 and 11 other sarbecoviruses. For these receptor-binding domains we determine thermal resilience (for mosaic vaccine development) and cross-recognition by antibodies. Antibody EY6A reacts across all tested sarbecoviruses, towards potential application against new coronavirus pandemic threats.

SUBMITTER: Vester SK

PROVIDER: S-EPMC9240080 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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SpySwitch enables pH- or heat-responsive capture and release for plug-and-display nanoassembly.

Vester Susan K SK Rahikainen Rolle R Khairil Anuar Irsyad N A INA Hills Rory A RA Tan Tiong Kit TK Howarth Mark M

Nature communications 20220628 1

Proteins can be empowered via SpyTag for anchoring and nanoassembly, through covalent bonding to SpyCatcher partners. Here we generate a switchable version of SpyCatcher, allowing gentle purification of SpyTagged proteins. We introduce numerous histidines adjacent to SpyTag's binding site, giving moderate pH-dependent release. After phage-based selection, our final SpySwitch allows purification of SpyTag- and SpyTag003-fusions from bacterial or mammalian culture by capture at neutral pH and rele ...[more]

PMID: 35764623

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Project description:Doxorubicin is a chemotherapeutic drug typically administered systemically which frequently leads to cardiac and hepatic toxicities. Local delivery to a tumor has a chance to mitigate some of these toxicities and can further be mitigated by including a means of tumor-specific drug release. Our laboratory has explored the use of molecular interactions to control the rate of drug release beyond that capable of diffusion alone. To this system, we added an additional affinity group (adamantane) to doxorubicin through a pH-sensitive hydrazone bond. The result was a modified doxorubicin which had an even higher affinity to our drug delivery polymer, and virtually no release in normal conditions, but showed accelerated release of drug in tumor-like low pH. Further, we show that adamantane-modified doxorubicin (adamantane-doxorubicin) and cleaved adamantane-doxorubicin showed equivalent capacity to kill human U-87 glioblastoma cells in vitro as unmodified doxorubicin. Taken together, these data demonstrate our ability to load high levels of modified chemotherapeutic drugs into our affinity-based delivery platform and deliver these drugs almost exclusively in the acidic microenvironments, such as those surrounding the tumor tissue via pH-cleavable bond while minimizing drug delivery in neutral pH tissue, with the ultimate goal of reducing systemic through better local delivery. Impact statement Doxorubicin (DOX) is especially cytotoxic to the heart, liver, kidneys, and healthy tissues surrounding the tumor microenvironment. This systemic toxicity can be partially addressed by local, tumor-specific drug delivery systems. While pH-sensitive DOX delivery systems have been developed by several other groups, many lack a prolonged and consistent release profile required to successfully treat heterogeneous tumors. Our system of a chemically modified form of DOX combined with an affinity-based cyclodextrin delivery system is capable of delivering DOX for 87 days while maintaining its the drug cytotoxicity. This finding is particularly relevant to improving cancer treatments because it enables regulated local delivery of DOX specifically to tumor tissue and allows the drug to be continuously delivered over a therapeutically relevant amount of time.

Dataset Information

SpySwitch enables pH- or heat-responsive capture and release for plug-and-display nanoassembly.

Publications

SpySwitch enables pH- or heat-responsive capture and release for plug-and-display nanoassembly.

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