Sequence-based prediction of permissive stretches for internal protein tagging and knockdown.
Sequence-based prediction of permissive stretches for internal protein tagging and knockdown.
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DOI：
10.1186/s12915-017-0440-0
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发表时间：
2017-10-30
期刊：
影响因子：
5.4
通讯作者：
Billerbeck S
中科院分区：
文献类型：
--
作者：
Oesterle S;Roberts TM;Widmer LA;Mustafa H;Panke S;Billerbeck S
通过将小功能肽插入表面可及的允许位点来对蛋白质进行内部标记已被证明是基础科学和应用科学不可或缺的工具。允许位点通常通过转座子诱变在个案基础上鉴定，限制了可扩展性及其作为全系统蛋白质工程工具的开发。我们开发了一种基于同源蛋白质中长度可变区（包含indel的区域）的识别来预测蛋白质中的容许延伸（PS）的方法。我们验证了蛋白质的一级结构信息本身就足以识别PSs.Identified PS被预测为主要是表面可访问的，因此，插入肽的位置可能适合于不同的应用。我们证明了这种方法的可行性，通过插入烟草蚀纹病毒蛋白酶识别位点（TEV标签）到几个PS在广泛的蛋白质，从小单体酶（腺苷酸激酶）到大的多亚基分子机器（ATP合成酶），并验证其插入后的功能。我们应用这种方法直接在大肠杆菌染色体中设计条件性蛋白质敲低，并产生具有增强的核苷酸稳定性的无细胞平台。功能性内部标记蛋白质可以合理设计并直接在染色体上实现。成功设计蛋白质敲除的关键是结合表面可及性和二级结构预测，以及设计改进的TEV标签，当插入蛋白质中间时能够有效水解。这种通用和便携的方法可能适用于其他应用，并被广泛采用。我们提供了内部标记蛋白质的设计指南，以使很少或没有蛋白质工程专业知识的科学家能够在内部标记他们的目标蛋白质。本文的在线版本（doi：10.1186/s12915 - 017 - 0440 - 0）包含补充材料，可供授权用户使用。
Internal tagging of proteins by inserting small functional peptides into surface accessible permissive sites has proven to be an indispensable tool for basic and applied science. Permissive sites are typically identified by transposon mutagenesis on a case-by-case basis, limiting scalability and their exploitation as a system-wide protein engineering tool. We developed an apporach for predicting permissive stretches (PSs) in proteins based on the identification of length-variable regions (regions containing indels) in homologous proteins. We verify that a protein's primary structure information alone is sufficient to identify PSs. Identified PSs are predicted to be predominantly surface accessible; hence, the position of inserted peptides is likely suitable for diverse applications. We demonstrate the viability of this approach by inserting a Tobacco etch virus protease recognition site (TEV-tag) into several PSs in a wide range of proteins, from small monomeric enzymes (adenylate kinase) to large multi-subunit molecular machines (ATP synthase) and verify their functionality after insertion. We apply this method to engineer conditional protein knockdowns directly in the Escherichia coli chromosome and generate a cell-free platform with enhanced nucleotide stability. Functional internally tagged proteins can be rationally designed and directly chromosomally implemented. Critical for the successful design of protein knockdowns was the incorporation of surface accessibility and secondary structure predictions, as well as the design of an improved TEV-tag that enables efficient hydrolysis when inserted into the middle of a protein. This versatile and portable approach can likely be adapted for other applications, and broadly adopted. We provide guidelines for the design of internally tagged proteins in order to empower scientists with little or no protein engineering expertise to internally tag their target proteins. The online version of this article (doi:10.1186/s12915-017-0440-0) contains supplementary material, which is available to authorized users.
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影响因子：
6.4
作者：
Billerbeck S;Panke S
通讯作者：
Panke S
影响因子：
15
作者：
Barthelmes, Katja;Reynolds, Anne M.;Peisach, Ezra;Jonker, Hendrik R. A.;DeNunzio, Nicholas J.;Allen, Karen N.;Imperiali, Barbara;Schwalbe, Harald
通讯作者：
Schwalbe, Harald
影响因子：
48
作者：
Chen, I;Howarth, M;Ting, AY
通讯作者：
Ting, AY
影响因子：
14.9
作者：
Bonde MT;Klausen MS;Anderson MV;Wallin AI;Wang HH;Sommer MO
通讯作者：
Sommer MO
影响因子：
3.2
作者：
HSU, CC;FOX, CF
通讯作者：
FOX, CF