A Helping Hand to Overcome Solubility Challenges in Chemical Protein Synthesis.
A Helping Hand to Overcome Solubility Challenges in Chemical Protein Synthesis.
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DOI：
10.1021/jacs.6b05719
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发表时间：
2016-09-14
影响因子：
15
通讯作者：
Kay MS
中科院分区：
文献类型：
--
作者：
Jacobsen MT;Petersen ME;Ye X;Galibert M;Lorimer GH;Aucagne V;Kay MS
虽然天然化学连接（NCL）和相关的化学选择性连接方法提供了将未保护的肽缝合在一起的优雅方法，但是不溶性和易于聚集的肽和组装中间体的处理和纯化产生了通过完全合成手段常规制备大蛋白质的瓶颈。在这项工作中，我们介绍了一种通用的新工具，Fmoc-Ddae-OH，N-Fmoc-1-（4，4-二甲基-2，6-二氧代环己基）-3-[2-（2-氨基乙氧基）乙氧基]-丙-1-醇，一种异双功能的无痕连接子，用于临时连接高度增溶的肽序列（帮手）到不溶性肽上。该工具以三个简单且几乎定量的步骤实施：（i）在Lys残基ε-胺处的连接体的树脂上掺入，（ii）所需增溶序列的Fmoc-SPPS延伸，以及（iii）在基于NCL的组装后，使用温和的含水肼在溶液中去除增溶序列以切割Ddae连接体。成功引入和去除Lys 6帮手首先在两个模型系统（埃博拉病毒C20肽和70个残基的核糖体蛋白L31）中得到证实。然后将其应用于具有挑战性的97个残基的辅助分子伴侣GroES的化学合成，GroES含有高度不溶性的C-末端片段，该片段被帮助之手拯救。重要的是，Ddae接头可以在NCL或脱硫后在一锅中裂解。相对于重组对照，验证了合成L-GroES的纯度、结构和分子伴侣活性。此外，辅助之手能够合成D-GroES，其在与重组GroEL的异手性混合物中是无活性的-提供了对伴侣特异性的额外了解。最终，这种简单，强大，易于使用的工具预计将广泛适用于具有挑战性的肽和蛋白质的合成。
Although native chemical ligation (NCL) and related chemoselective ligation approaches provide an elegant method to stitch together unprotected peptides, the handling and purification of insoluble and aggregation-prone peptides and assembly intermediates create a bottleneck to routinely preparing large proteins by completely synthetic means. In this work, we introduce a general new tool, Fmoc-Ddae-OH, N-Fmoc-1-(4,4-dimethyl-2,6-dioxocyclo-hexylidene)-3-[2-(2-aminoethoxy)ethoxy]-propan-1-ol, a hetero-bifunctional traceless linker for temporarily attaching highly solubilizing peptide sequences (helping hands) onto insoluble peptides. This tool is implemented in three simple and nearly quantitative steps: (i) on-resin incorporation of the linker at a Lys residue ε-amine, (ii) Fmoc-SPPS elongation of a desired solubilizing sequence, and (iii) in-solution removal of the solubilizing sequence using mild aqueous hydrazine to cleave the Ddae linker after NCL-based assembly. Successful introduction and removal of a Lys6 helping hand is first demonstrated in two model systems (Ebolavirus C20 peptide and the 70-residue ribosomal protein L31). It is then applied to the challenging chemical synthesis of the 97-residue co-chaperonin GroES, which contains a highly insoluble C-terminal segment that is rescued by a helping hand. Importantly, the Ddae linker can be cleaved in one pot following NCL or desulfurization. The purity, structure, and chaperone activity of synthetic L-GroES were validated with respect to a recombinant control. Additionally, the helping hand enabled synthesis of D-GroES, which was inactive in a hetero-chiral mixture with recombinant GroEL—providing additional insight into chaperone specificity. Ultimately, this simple, robust, and easy-to-use tool is expected to be broadly applicable for the synthesis of challenging peptides and proteins.
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