Sneaking in SpyCatcher using cell penetrating peptides forin vivoimaging.
Sneaking in SpyCatcher using cell penetrating peptides forin vivoimaging.
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DOI：
10.1088/1361-6528/acdf65
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发表时间：
2023-08-02
期刊：
影响因子：
3.5
通讯作者：
中科院分区：
文献类型：
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蛋白质复合物的体内成像是了解这些关键生物分子的潜在生物学功能的有力方法。尽管小的、高亲和力的纳米抗体的工程化已经变得更加普遍，但是这些标签的解离速率可能导致活细胞中蛋白质的不完全或部分标记。SpyCatcher 003和SpyTag分裂蛋白系统允许与短靶肽不可逆地共价结合，这与基于纳米抗体亲和力的探针不同。然而，将这些标签输送到细胞中而不破坏其正常功能是一个关键挑战。细胞穿透肽（CPP）是短的肽序列，其促进膜不可渗透的“货物”（例如蛋白质）转导到细胞中。在这里，我们报告我们的努力，工程师和特点CPP-SpyCatcher 003融合模块化成像探头。我们选择了三个CPP，CUPID，Pentratin，和pVEC，工程融合蛋白探针的超分辨率显微镜，目的是消除先前的透化处理，可能会引入成像伪影。我们发现，融合的CPP序列SpyCatcher 003导致二聚体和多聚体的形成，确定通过尺寸排阻色谱法，动态光散射，和抗SDS二聚体的SDS-PAGE凝胶。通过分离和标记工程蛋白的单体形式，我们表明这些构建体保留了其结合SpyTag的能力，并且所有三个CPP序列都保持膜活性，这在SDS去污剂存在下通过CD光谱法进行了评估。使用荧光和超分辨率晶格结构照明显微镜（Lattice SIM）成像，我们表明CPP没有增强E.然而，对于新月柄杆菌细胞，我们表明Penetratin和较小程度的CUPID确实增强了摄取。我们的研究结果证明了CPP-SpyCatcher 003在活细胞内标记靶点的能力，为使用分裂蛋白系统进行靶向体内成像提供了基础。
In vivo imaging of protein complexes is a powerful method for understanding the underlying biological function of these key biomolecules. Though the engineering of small, high affinity nanobodies have become more prevalent, the off-rates of these tags may result in incomplete or partial labeling of proteins in live cells. The SpyCatcher003 and SpyTag split protein system allow for irreversible, covalent binding to a short target peptide unlike nanobody-affinity based probes. However, delivering these tags into a cell without disrupting its normal function is a key challenge. Cell penetrating peptides (CPPs) are short peptide sequences that facilitate the transduction of otherwise membrane-impermeable ‘cargo’ , such as proteins, into cells. Here we report on our efforts to engineer and characterize CPP-SpyCatcher003 fusions as modular imaging probes. We selected three CPPs, CUPID, Pentratin, and pVEC, to engineer fusion protein probes for superresolution microscopy, with the aim to eliminate prior permeabilization treatments that could introduce imaging artifacts. We find that fusing the CPP sequences to SpyCatcher003 resulted in dimer and multimer formation as determined by size exclusion chromatography, dynamic light scattering, and SDS resistant dimers on SDS-PAGE gels. By isolating and labeling the monomeric forms of the engineered protein, we show these constructs retained their ability to bind SpyTag and all three CPP sequences remain membrane active, as assessed by CD spectroscopy in the presence of SDS detergent. Using fluorescence and super resolution Lattice structured illumination microscopy (Lattice SIM) imaging we show that the CPPs did not enhance uptake of SpyCatcher by E. coli, however with Caulobacter crescentus cells, we show that Penetratin, and to a lesser degree CUPID, does enhance uptake. Our results demonstrate the ability of the CPP-SpyCatcher003 to label targets within living cells, providing the groundwork for using split protein systems for targeted in vivo imaging.
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影响因子：
17.1
作者：
Bruun TUJ;Andersson AC;Draper SJ;Howarth M
通讯作者：
Howarth M
影响因子：
48
作者：
Grimm, Jonathan B.;English, Brian P.;Lavis, Luke D.
通讯作者：
Lavis, Luke D.
影响因子：
4.8
作者：
Derossi, D;Calvet, S;Prochiantz, A
通讯作者：
Prochiantz, A
影响因子：
5.5
作者：
Avci FG;Akbulut BS;Ozkirimli E
通讯作者：
Ozkirimli E
DOI：
10.3390/molecules22111929
发表时间：
2017-11-08
期刊：
Molecules (Basel, Switzerland)
影响因子：
--
作者：
Kalafatovic D;Giralt E
通讯作者：
Giralt E