SpySwitch enables pH- or heat-responsive capture and release for plug-and-display nanoassembly.
SpySwitch enables pH- or heat-responsive capture and release for plug-and-display nanoassembly.
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DOI：
10.1038/s41467-022-31193-8
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发表时间：
2022-06-28
影响因子：
16.6
通讯作者：
中科院分区：
文献类型：
--
作者：
蛋白质可以通过SpyTag进行锚定和纳米组装，通过共价键合到SpyCatcher合作伙伴。在这里，我们生成了SpyCatcher的可切换版本，允许温和纯化SpyTagged蛋白质。我们在SpyTag的结合位点附近引入许多组氨酸，从而产生适度的pH依赖性释放。在基于噬菌体的选择之后，我们的最终SpySwitch允许通过在中性pH下捕获并在pH 5下释放来从细菌或哺乳动物培养物中纯化SpyTag-和SpyTag 003-融合物，其纯度远远超过His-标签方法。SpySwitch也是热敏的，在4 °C捕获并在37 °C释放。通过灵活选择洗脱液，SpySwitch纯化的蛋白质可以直接组装到多聚体支架上。60-mer多聚化增强了免疫原性，我们使用SpySwitch从SARS-CoV-2和其他11种肉瘤病毒中纯化受体结合结构域。对于这些受体结合域，我们确定热弹性（嵌合疫苗开发）和抗体的交叉识别。抗体EY 6A对所有测试的Sarbecoviruses都有反应，具有对抗新型冠状病毒大流行威胁的潜在应用。SpyCatcher-SpyTag系统允许蛋白质锚定和纳米组装。在这里，作者设计了SpySwitch，这是一种双重可切换的Catcher，可以在下游应用（如病毒样颗粒的组装）之前温和地纯化SpyTagged蛋白质。
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. The SpyCatcher-SpyTag system allows protein anchoring and nanoassembly. Here, the authors engineer SpySwitch, a dually switchable Catcher which allows gentle purification of SpyTagged proteins prior to downstream applications such as the assembly of virus-like particles.
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影响因子：
7.3
作者：
Brune KD;Howarth M
通讯作者：
Howarth M
影响因子：
64.8
作者：
Barnes CO;Jette CA;Abernathy ME;Dam KA;Esswein SR;Gristick HB;Malyutin AG;Sharaf NG;Huey-Tubman KE;Lee YE;Robbiani DF;Nussenzweig MC;West AP Jr;Bjorkman PJ
通讯作者：
Bjorkman PJ
影响因子：
17.1
作者：
Bruun TUJ;Andersson AC;Draper SJ;Howarth M
通讯作者：
Howarth M
影响因子：
2.7
作者：
Armanini, D;Nacamulli, D;Fiore, C
通讯作者：
Fiore, C
DOI：
10.1073/pnas.1510199112
发表时间：
2015-08-18
影响因子：
11.1
作者：
Corti, Davide;Zhao, Jincun;Lanzavecchia, Antonio
通讯作者：
Lanzavecchia, Antonio