Controlling Macromolecular Topology with Genetically Encoded SpyTag鈥揝pyCatcher Chemistry

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• 作者：Wen-Bin Zhang ; Fei Sun ; David A. Tirrell ; Frances H. Arnold
• 刊名：Journal of the American Chemical Society
• 出版年：2013
• 出版时间：September 18, 2013
• 年：2013
• 卷：135
• 期：37
• 页码：13988-13997
• 全文大小：587K
• 年卷期：v.135,no.37(September 18, 2013)
• ISSN：1520-5126

文摘

Control of molecular topology constitutes a fundamental challenge in macromolecular chemistry. Here we describe the synthesis and characterization of artificial elastin-like proteins (ELPs) with unconventional nonlinear topologies including circular, tadpole, star, and H-shaped proteins using genetically encoded SpyTag鈥揝pyCatcher chemistry. SpyTag is a short polypeptide that binds its protein partner SpyCatcher and forms isopeptide bonds under physiological conditions. Sequences encoding SpyTag and SpyCatcher can be strategically placed into ELP genes to direct post-translational topological modification in situ. Placement of SpyTag at the N-terminus and SpyCatcher at the C-terminus directs formation of circular ELPs. Induction of expression at 16 掳C with 10 渭M IPTG yields 80% monomeric cyclic protein. When SpyTag is placed in the middle of the chain, it exhibits an even stronger tendency toward cyclization, yielding up to 94% monomeric tadpole proteins. Telechelic ELPs containing either SpyTag or SpyCatcher can be expressed, purified, and then coupled spontaneously upon mixing in vitro. Block proteins, 3-arm or 4-arm star proteins, and H-shaped proteins have been prepared, with the folded CnaB2 domain that results from the SpyTag鈥揝pyCatcher reaction as the molecular core or branch junction. The modular character of the SpyTag鈥揝pyCatcher strategy should make it useful for preparing nonlinear macromolecules of diverse sequence and structure.