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首页> 外文期刊>The FEBS journal >The competitor-introduced Gd recruitment system, a new approach for screening affinity-enhanced proteins
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The competitor-introduced Gd recruitment system, a new approach for screening affinity-enhanced proteins

机译：竞争对手引入的Gd募集系统，一种筛选亲和力增强蛋白的新方法

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We have developed a new approach based on the Gd recruitment system to screen affinity-enhanced proteins by expressing a binding competitor. The previously established Gd recruitment system is a yeast two-hybrid (Y2H) system that utilizes G-protein signaling, and is based on the fact that membrane localization of the G-protein d subunit (Gd) is essential for signal transduction in yeast. In the original Y2H system, an engineered Gd that lacks membrane localization upon deletion of the lipid modification site (Gdcyto) is produced, and a candidate protein with an artificial lipidation site and its counterpart fused with Gdcyto are expressed. As protein-protein interactions bring Gdcyto towards the plasma membrane, G-protein signaling can be activated, and the interaction is detected by various cellular responses as the readout. In the current study, we expressed a third cytosolic protein that competes with the candidate protein to specifically isolate affinity-enhanced mutants from a mutation library of the candidate protein. Enhancing the affinity of the protein candidate guides the counterpart-Gdcyto fusion protein towards the plasma membrane and activates signaling. Using mutants of the Z domain derived from Staphylococcus aureus protein A as candidate proteins or competitors, and the Fc portion of human immunoglobulin G (IgG) as the counterpart, we demonstrate that affinity-enhanced proteins can be effectively screened from a library containing a 10 000-fold excess of non-enhanced proteins. This new approach, called the competitor-introduced Gd recruitment system, will be useful for efficient discovery of rare valuable candidates hidden among excess ordinary ones. Structured digital abstract MINT-7556266: Fc portion of human IgG (uniprotkb: P01857) physically interacts ( MI:0915) with Z domain of protein A (uniprotkb: P38507) by two hybrid ( MI:0018)
机译：我们已经开发了一种基于Gd募集系统的新方法，可以通过表达结合竞争对手来筛选亲和力增强的蛋白质。先前建立的Gd募集系统是利用G蛋白信号转导的酵母双杂交（Y2H）系统，基于以下事实：G蛋白d亚基（Gd）的膜定位对于酵母中的信号转导至关重要。在最初的Y2H系统中，产生了在缺失脂质修饰位点（Gdcyto）时缺乏膜定位的工程化Gd，并表达了具有人工脂质化位点的候选蛋白及其与Gdcyto融合的对应蛋白。当蛋白质-蛋白质相互作用将Gdcyto移向质膜时，G-蛋白质信号传导可以被激活，并且通过各种细胞反应作为读数来检测相互作用。在当前的研究中，我们表达了与候选蛋白竞争的第三种胞质蛋白，可从候选蛋白的突变文库中特异性分离亲和力增强的突变体。增强蛋白质候选物的亲和力将对应的Gd细胞融合蛋白导向质膜并激活信号传导。使用源自金黄色葡萄球菌蛋白A的Z结构域的突变体作为候选蛋白或竞争者，并使用人类免疫球蛋白G（IgG）的Fc部分作为对应物，我们证明可以从包含10个分子的文库中有效筛选亲和力增强的蛋白非增强蛋白过量000倍。这种称为竞争对手引入的Gd招聘系统的新方法，对于有效发现隐藏在超凡普通候选人中的稀有有价值的候选人将很有用。结构化数字摘要MINT-7556266：人IgG（uniprotkb：P01857）的Fc部分与蛋白质A（uniprotkb：P38507）的Z结构域通过两个杂交（MI：0018）物理相互作用（MI：0915）

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