我们最近报道了一种亲和定向的蛋白质导弹(AdPROM)系统,用于目标内源性蛋白质(POI)的靶向蛋白水解(Fulcher等人。,2016年和2017年)。AdPROM由VonHippelLindau(VHL)蛋白组成,aCullin2E3连接酶底物受体(Bosu和Kipreos,2008),缀合至识别细胞中的靶蛋白的高亲和力多肽结合剂(例如骆驼科纳米抗体)。当引入细胞时,靶蛋白被募集到CUL2E3泛素连接酶复合物中,用于泛素介导的蛋白酶体降解.对于目标蛋白募集,我们已经利用了骆驼衍生的VHH结构域纳米抗体以及基于人III型纤连蛋白结构域的合成多肽单体(Sha等人。,2013;Fridy等人。,2014年;施密特等人。,2016)。在这个协议中,我们描述了产生AdPROM构建体的详细方法及其在人细胞系中用于靶蛋白破坏的应用。AdPROM允许POI的功能表征及其靶蛋白破坏的效率克服了RNA干扰方法的许多限制。这需要长时间的治疗,并与脱靶效应有关,和CRISPR/Cas9基因编辑,这并不总是可行的。
We recently reported an Affinity-directed PROtein Missile (
AdPROM) system for the targeted proteolysis of endogenous proteins of interest (POI) ( Fulcher et al., 2016 and 2017).
AdPROM consists of the Von Hippel Lindau (VHL) protein, a Cullin 2 E3 ligase substrate receptor (Bosu and Kipreos, 2008), conjugated to a high affinity polypeptide binder (such as a camelid nanobody) that recognises the target protein in cells. When introduced in cells, the target protein is recruited to the CUL2 E3 ubiquitin ligase complex for ubiquitin-mediated proteasomal degradation. For target protein recruitment, we have utilised both camelid-derived VHH domain nanobodies as well as synthetic polypeptide monobodies based on the human type III fibronectin domain ( Sha et al., 2013 ; Fridy et al., 2014 ; Schmidt et al., 2016 ). In this protocol, we describe detailed methodology involved in generating
AdPROM constructs and their application in human cell lines for target protein destruction.
AdPROM allows functional characterisation of the POI and its efficiency of target protein destruction overcomes many limitations of RNA-interference approaches, which necessitate long treatments and are associated with off-target effects, and CRISPR/Cas9 gene editing, which is not always feasible.