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 any Strep-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 bound Strep-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.

中文翻译：

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异双功能生物聚合物的工程设计，用于 Strep-Tag 蛋白和病毒样纳米颗粒的可调结合和沉淀


亲和沉淀是一种强大的分离方法，因为它将亲和层析的结合选择性与通过环境（如 pH、离子强度、温度、光线等）的微小变化触发的相分离来沉淀捕获的生物分子相结合。弹性蛋白样多肽 （ELP） 是由五肽重复序列 VPGVG 组成的热响应性生物聚合物，经过可逆相分离，当温度和/或盐浓度升高时，它们会聚集。在这里，我们描述了与可溶性链霉亲和素突变体的 ELP 融合的产生，该突变体能够快速纯化任何感兴趣的 Strep-tag II 融合蛋白。这种异双功能蛋白利用链霉亲和素的天然四聚体结构，在蛋白质捕获时产生结合诱导的多价交联。结合的 Strep-tag II 融合蛋白与链霉亲和素-ELP 捕获支架的生物素介导的高效解离可实现温和的洗脱条件。我们还表明，该平台在纯化病毒样颗粒 （VLP） 样 E2 蛋白纳米颗粒方面特别有效，这可能是因为蛋白质颗粒的高化合价导致结合诱导的交联和沉淀。考虑到 VLP 对基因治疗应用的重要性，我们认为这是一个特别令人兴奋的进步。我们通过高效纯化 VLP 样 E2 蛋白纳米颗粒作为替代物来证明这种可行性。