Supramolecular assembly of multifunctional protein gels via an N-glycosylation consensus sequence fusion domain†

IF 3.2 3区 工程技术 Q2 CHEMISTRY, PHYSICAL Molecular Systems Design & Engineering Pub Date : 2024-05-24 DOI:10.1039/D4ME00029C
Eric D. Hill, Stephen Michel, Natasha R. Sequeira, Benjamin G. Keselowsky and Gregory A. Hudalla
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Abstract

Polypeptide fusion tags that can direct the assembly of folded proteins into supramolecular networks are attractive for creating functional biomaterials. A practical challenge is identifying polypeptide sequences that form supramolecular networks in response to specific user-controlled stimuli, which is advantageous for producing polypeptide–protein fusions using cell-based expression hosts. Here, we report an N-glycosylation tag, (GGGSGGGSGGNWTT)10 or “NGT,” that assembles into a supramolecular network at reduced temperatures when fused to a folded protein. For example, NGT fused to superfolder green fluorescent protein (NGTsfGFP) formed materials that emitted green fluorescence in blue light, while NGT fused to NanoLuc luciferase (NGTnL) formed materials that emitted blue light in the presence of the chemical substrate furimazine. Oscillatory rheology established the materials as weak viscoelastic gels that can undergo shear-thinning and self-healing. Gel formation could be disrupted by mutating the asparagines in NGT to glutamines, introducing a chaotropic agent, or modifying the asparagines in NGT with glucose, suggesting a role for hydrogen bonds involving asparagine in supramolecular network formation. A mixture of soluble NGTsfGFP and NGTnL formed a multifunctional gel at reduced temperature that demonstrated bioluminescence resonance energy transfer between the nL and sfGFP domains in the presence of furimazine. Collectively, these data establish NGT as a temperature-responsive polypeptide tag that can be used to create functional biomaterials from soluble fusion proteins synthesized by cell-based hosts.

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通过 N-糖基化共识序列融合域超分子组装多功能蛋白质凝胶
能引导折叠蛋白质组装成超分子网络的多肽融合标签对创造功能性生物材料很有吸引力。一个实际的挑战是识别能在用户控制的特定刺激下形成超分子网络的多肽序列,这对利用基于细胞的表达宿主生产多肽-蛋白质融合体是有利的。在这里,我们报告了一种 N-糖基化标签 (GGGSGGSGGGNWTT)10 或 "NGT",当它与折叠蛋白融合时,可在较低温度下组装成超分子网络。例如,与超级折叠绿色荧光蛋白(NGTsfGFP)融合的 NGT 形成的材料在蓝光下发出绿色荧光,而与 NanoLuc 荧光素酶(NGTnL)融合的 NGT 形成的材料在化学底物呋喃嗪的存在下发出蓝光。振荡流变学确定了这些材料是弱粘弹性凝胶,可以发生剪切稀化和自我修复。通过将 NGT 中的天冬酰胺突变为谷氨酸、引入混沌剂或用葡萄糖修饰 NGT 中的天冬酰胺,可以破坏凝胶的形成。可溶性 NGTsfGFP 和 NGTnL 的混合物在低温下形成了多功能凝胶,在呋喃嗪的存在下,nL 和 sfGFP 结构域之间发生了生物发光共振能量转移。总之,这些数据证明 NGT 是一种温度响应性多肽标签,可用于利用细胞宿主合成的可溶性融合蛋白制造功能性生物材料。
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来源期刊
Molecular Systems Design & Engineering
Molecular Systems Design & Engineering Engineering-Biomedical Engineering
CiteScore
6.40
自引率
2.80%
发文量
144
期刊介绍: Molecular Systems Design & Engineering provides a hub for cutting-edge research into how understanding of molecular properties, behaviour and interactions can be used to design and assemble better materials, systems, and processes to achieve specific functions. These may have applications of technological significance and help address global challenges.
期刊最新文献
Back cover Back cover Dual responsive fluorescence switching of organohydrogel towards base/acid† Back cover Graph-based networks for accurate prediction of ground and excited state molecular properties from minimal features†
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