Takeda Hiroyuki, Zhou Wei, Kido Kohki, Suno Ryoji, Iwasaki Takahiro, Kobayashi Takuya, Sawasaki Tatsuya
Proteo-Science Center, Ehime University, Matsuyama, Ehime, Japan.
Department of Medical Chemistry and Cell Biology, Kyoto University Graduate School of Medicine, Yoshida, Sakyo-ku, Kyoto, Japan.
PLoS One. 2017 May 25;12(5):e0178246. doi: 10.1371/journal.pone.0178246. eCollection 2017.
There are many strategies to purify recombinant proteins of interest, and affinity purification utilizing monoclonal antibody that targets a linear epitope sequence is one of the essential techniques used in current biochemistry and structural biology. Here we introduce a new protein purification system using a very short CP5 tag. First, we selected anti-dopamine receptor D1 (DRD1) rabbit monoclonal antibody clone Ra62 (Ra62 antibody) as capture antibody, and identified its minimal epitope sequence as a 5-amino-acid sequence at C-terminal of DRD1 (GQHPT-COOH, D1CE sequence). We found that single amino acid substitution in D1CE sequence (GQHVT-COOH) increased dissociation rate up to 10-fold, and named the designed epitope sequence CP5 tag. Using Ra62 antibody and 2 peptides with different affinity, we developed a new affinity protein purification method, CP5 system. Ra62 antibody quickly captures CP5-tagged target protein, and captured CP5-tagged protein was eluted by competing with higher affinity D1CE peptide. By taking the difference of the affinity between D1CE and CP5, sharp elution under mild condition was achieved. Using CP5 system, we successfully purified deubiquitinase CYLD and E3 ubiquitin ligase MARCH3, and detected their catalytic activity. As to G protein-coupled receptors (GPCRs), 9 out of 12 cell-free synthesized ones were purified, demonstrating its purification capability of integral membrane proteins. CP5 tagged CHRM2 expressed by baculovirus-insect cell was also successfully purified by CP5 system. CP5 system offers several distinct advantages in addition to its specificity and elution performance. CP5 tag is easy to construct and handle because of its short length, which has less effect on protein characters. Mild elution of CP5 system is particulaly suitable for preparing delicate proteins such as enzymes and membrane proteins. Our data demonstrate that CP5 system provides a new promising option in protein sample preparation with high yield, purity and activity for downstream applications in functional and structural analysis.
纯化目标重组蛋白有多种策略，利用靶向线性表位序列的单克隆抗体进行亲和纯化是当前生物化学和结构生物学中使用的关键技术之一。在此，我们介绍一种使用非常短的CP5标签的新型蛋白质纯化系统。首先，我们选择抗多巴胺受体D1（DRD1）兔单克隆抗体克隆Ra62（Ra62抗体）作为捕获抗体，并确定其最小表位序列为DRD1 C末端的一个5氨基酸序列（GQHPT-COOH，D1CE序列）。我们发现D1CE序列中的单个氨基酸取代（GQHVT-COOH）可使解离速率提高至10倍，并将设计的表位序列命名为CP5标签。使用Ra62抗体和两种具有不同亲和力的肽，我们开发了一种新的亲和蛋白纯化方法，即CP5系统。Ra62抗体可快速捕获带有CP5标签的目标蛋白，而捕获的带有CP5标签的蛋白通过与更高亲和力的D1CE肽竞争而被洗脱。通过利用D1CE和CP5之间亲和力的差异，在温和条件下实现了尖锐洗脱。使用CP5系统，我们成功纯化了去泛素化酶CYLD和E3泛素连接酶MARCH3，并检测了它们的催化活性。对于G蛋白偶联受体（GPCR），12个无细胞合成的受体中有9个被纯化，证明了其对整合膜蛋白的纯化能力。杆状病毒-昆虫细胞表达的带有CP5标签的毒蕈碱型乙酰胆碱受体M2（CHRM2）也通过CP5系统成功纯化。CP5系统除了具有特异性和洗脱性能外，还具有几个明显的优点。CP5标签因其长度短而易于构建和操作，对蛋白质特性的影响较小。CP5系统的温和洗脱特别适合制备诸如酶和膜蛋白等精细蛋白质。我们的数据表明，CP5系统在蛋白质样品制备方面提供了一个新的有前景的选择，可为功能和结构分析的下游应用提供高产率、高纯度和高活性的蛋白质。
