Huet Simon, Gorre Harmony, Perrocheau Anaëlle, Picot Justine, Cinier Mathieu
Affilogic SAS, Nantes, France.
PLoS One. 2015 Nov 5;10(11):e0142304. doi: 10.1371/journal.pone.0142304. eCollection 2015.
With the continuous diversification of recombinant DNA technologies, the possibilities for new tailor-made protein engineering have extended on an on-going basis. Among these strategies, the use of the green fluorescent protein (GFP) as a fusion domain has been widely adopted for cellular imaging and protein localization. Following the lead of the direct head-to-tail fusion of GFP, we proposed to provide additional features to recombinant proteins by genetic fusion of artificially derived binders. Thus, we reported a GFP-ready fusion tag consisting of a small and robust fusion-friendly anti-GFP Nanofitin binding domain as a proof-of-concept. While limiting steric effects on the carrier, the GFP-ready tag allows the capture of GFP or its blue (BFP), cyan (CFP) and yellow (YFP) alternatives. Here, we described the generation of the GFP-ready tag from the selection of a Nanofitin variant binding to the GFP and its spectral variants with a nanomolar affinity, while displaying a remarkable folding stability, as demonstrated by its full resistance upon thermal sterilization process or the full chemical synthesis of Nanofitins. To illustrate the potential of the Nanofitin-based tag as a fusion partner, we compared the expression level in Escherichia coli and activity profile of recombinant human tumor necrosis factor alpha (TNFα) constructs, fused to a SUMO or GFP-ready tag. Very similar expression levels were found with the two fusion technologies. Both domains of the GFP-ready tagged TNFα were proved fully active in ELISA and interferometry binding assays, allowing the simultaneous capture by an anti-TNFα antibody and binding to the GFP, and its spectral mutants. The GFP-ready tag was also shown inert in a L929 cell based assay, demonstrating the potent TNFα mediated apoptosis induction by the GFP-ready tagged TNFα. Eventually, we proposed the GFP-ready tag as a versatile capture and labeling system in addition to expected applications of anti-GFP Nanofitins (as illustrated with previously described state-of-the-art anti-GFP binders applied to living cells and in vitro applications). Through a single fusion domain, the GFP-ready tagged proteins benefit from subsequent customization within a wide range of fluorescence spectra upon indirect binding of a chosen GFP variant.
随着重组DNA技术的不断多样化，新型定制蛋白质工程的可能性一直在持续扩展。在这些策略中，绿色荧光蛋白（GFP）作为融合结构域已被广泛用于细胞成像和蛋白质定位。继GFP直接头对头融合的引领之后，我们提议通过人工衍生结合剂的基因融合为重组蛋白提供额外特性。因此，我们报道了一种由小型且坚固的融合友好型抗GFP纳米适配体结合结构域组成的GFP就绪融合标签作为概念验证。在限制对载体的空间位阻效应的同时，该GFP就绪标签能够捕获GFP或其蓝色（BFP）、青色（CFP）和黄色（YFP）变体。在此，我们描述了从与GFP及其光谱变体结合且具有纳摩尔亲和力的纳米适配体变体的选择中生成GFP就绪标签的过程，同时展示出显著的折叠稳定性，热灭菌过程中的完全抗性或纳米适配体的全化学合成证明了这一点。为了说明基于纳米适配体的标签作为融合伙伴的潜力，我们比较了与SUMO或GFP就绪标签融合的重组人肿瘤坏死因子α（TNFα）构建体在大肠杆菌中的表达水平和活性谱。两种融合技术的表达水平非常相似。GFP就绪标签的TNFα的两个结构域在ELISA和干涉测量结合试验中均被证明具有完全活性，能够同时被抗TNFα抗体捕获并与GFP及其光谱突变体结合。在基于L929细胞的试验中，GFP就绪标签也显示出惰性，证明了GFP就绪标签的TNFα具有强大的TNFα介导的凋亡诱导作用。最终，除了抗GFP纳米适配体的预期应用（如先前描述的应用于活细胞和体外应用的最先进抗GFP结合剂所示）之外，我们提议将GFP就绪标签作为一种通用的捕获和标记系统。通过单个融合结构域，GFP就绪标签的蛋白质在间接结合选定的GFP变体后，可在广泛的荧光光谱范围内受益于后续定制。
