Abstract：
Targeted protein degradation (TPD) is a useful approach in dissecting protein function and therapeutics. Technologies such as RNA interference or gene knockout that are routinely used rely on protein turnover. However, RNA interference takes a long time to deplete target proteins and is not suitable for long-lived proteins, while a genetic knockout is irreversible, takes a long time to achieve and is not suitable for essential genes. TPD has the potential to overcome the limitations of RNA interference and gene editing approaches. We have established the Affinity directed PROtein Missile (AdPROM) system, which harnesses nanobodies or binders of target proteins to redirect E3 ubiquitin ligase activity to the target protein to induce TPD through the ubiquitin proteasome system. Here we provide a step-by-step protocol for using the AdPROM system for targeted proteolysis of endogenously GFP-tagged K-RAS through an anti-GFP nanobody. This protocol can be amended to target a wide range of different proteins of interest (POIs) either by replacing the anti-GFP nanobody with a nanobody recognising the POI or by endogenously tagging the POI with GFP through CRISPR/Cas9 genome editing.
摘要：
靶向蛋白质降解（TPD）是解剖蛋白质功能和治疗的有用方法。常规使用的RNA干扰或基因敲除等技术依赖于蛋白质周转。然而,RNA干扰需要很长时间才能耗尽靶蛋白，并且不适合长寿命蛋白，虽然基因敲除是不可逆转的，需要很长时间才能实现，并且不适合必需基因。TPD有可能克服RNA干扰和基因编辑方法的局限性。我们已经建立了亲和定向PROtein导弹（AdPROM）系统，它利用目标蛋白的纳米抗体或结合剂将E3泛素连接酶活性重定向到目标蛋白，以通过泛素蛋白酶体系统诱导TPD。在这里，我们提供了使用AdPROM系统通过抗GFP纳米抗体靶向蛋白水解内源性GFP标记的K-RAS的逐步方案。可以通过用识别POI的纳米抗体替换抗GFP纳米抗体或通过CRISPR/Cas9基因组编辑用GFP内源性标记POI来修改该方案以靶向广泛的不同目的蛋白质(POI)。
