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Highly stable and efficient mRNA templates for mRNA-protein fusions and C-terminally labeled proteins.用于mRNA-蛋白质融合和C端标记蛋白质的高度稳定且高效的mRNA模板。
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2
Improvement of a puromycin-linker to extend the selection target varieties in cDNA display method.改良嘌呤霉素连接子以扩展 cDNA 展示方法中的选择靶标种类。
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Psoralen photo-crosslinked mRNA-puromycin conjugates: a novel template for the rapid and facile preparation of mRNA-protein fusions.补骨脂素光交联的mRNA-嘌呤霉素缀合物：一种快速简便制备mRNA-蛋白质融合体的新型模板。
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Solid-phase translation and RNA-protein fusion: a novel approach for folding quality control and direct immobilization of proteins using anchored mRNA.固相翻译与RNA-蛋白质融合：一种利用锚定mRNA进行蛋白质折叠质量控制和直接固定的新方法。
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In vitro selection of tRNAs for efficient four-base decoding to incorporate non-natural amino acids into proteins in an Escherichia coli cell-free translation system.在大肠杆菌无细胞翻译系统中进行tRNA的体外筛选，以实现高效四碱基解码从而将非天然氨基酸掺入蛋白质中。
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本文引用的文献

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Drug receptor identification from multiple tissues using cellular-derived mRNA display libraries.
Chem Biol. 2002 Jun;9(6):691-8. doi: 10.1016/s1074-5521(02)00148-5.
2
Novel fluorescence labeling and high-throughput assay technologies for in vitro analysis of protein interactions.用于蛋白质相互作用体外分析的新型荧光标记和高通量检测技术。
Genome Res. 2002 Mar;12(3):487-92. doi: 10.1101/gr.218802.
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Functional proteins from a random-sequence library.来自随机序列文库的功能蛋白。
Nature. 2001 Apr 5;410(6829):715-8. doi: 10.1038/35070613.
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In vitro selection and characterization of Bcl-X(L)-binding proteins from a mix of tissue-specific mRNA display libraries.
J Biol Chem. 2001 Jun 15;276(24):20898-906. doi: 10.1074/jbc.M011641200. Epub 2001 Mar 30.
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The use of mRNA display to select high-affinity protein-binding peptides.利用mRNA展示技术筛选高亲和力蛋白质结合肽。
Proc Natl Acad Sci U S A. 2001 Mar 27;98(7):3750-5. doi: 10.1073/pnas.061028198. Epub 2001 Mar 13.
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Selection of RNA-binding peptides using mRNA-peptide fusions.使用mRNA-肽融合体筛选RNA结合肽。
Methods. 2001 Mar;23(3):287-93. doi: 10.1006/meth.2000.1139.
7
Picomolar affinity antibodies from a fully synthetic naive library selected and evolved by ribosome display.通过核糖体展示从完全合成的天然文库中筛选和进化得到的皮摩尔亲和力抗体。
Nat Biotechnol. 2000 Dec;18(12):1287-92. doi: 10.1038/82407.
8
Psoralen photo-crosslinked mRNA-puromycin conjugates: a novel template for the rapid and facile preparation of mRNA-protein fusions.补骨脂素光交联的mRNA-嘌呤霉素缀合物：一种快速简便制备mRNA-蛋白质融合体的新型模板。
Nucleic Acids Res. 2000 Sep 15;28(18):E83. doi: 10.1093/nar/28.18.e83.
9
Optimized synthesis of RNA-protein fusions for in vitro protein selection.用于体外蛋白质筛选的RNA-蛋白质融合体的优化合成。
Methods Enzymol. 2000;318:268-93. doi: 10.1016/s0076-6879(00)18058-9.
10
Constructing high complexity synthetic libraries of long ORFs using in vitro selection.利用体外筛选构建长开放阅读框的高复杂性合成文库。
J Mol Biol. 2000 Mar 24;297(2):309-19. doi: 10.1006/jmbi.2000.3571.

用于mRNA-蛋白质融合和C端标记蛋白质的高度稳定且高效的mRNA模板。

Highly stable and efficient mRNA templates for mRNA-protein fusions and C-terminally labeled proteins.

作者信息

Miyamoto-Sato Etsuko, Takashima Hideaki, Fuse Shinichiro, Sue Kaori, Ishizaka Masamichi, Tateyama Seiji, Horisawa Kenichi, Sawasaki Tatsuya, Endo Yaeta, Yanagawa Hiroshi

机构信息

Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, 3-14-1, Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan.

出版信息

Nucleic Acids Res. 2003 Aug 1;31(15):e78. doi: 10.1093/nar/gng078.

DOI:10.1093/nar/gng078
PMID:12888530
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC169963/
Abstract

For high-throughput in vitro protein selection using genotype (mRNA)-phenotype (protein) fusion formation and C-terminal protein labeling as a post-selection analysis, it is important to improve the stability and efficiency of mRNA templates for both technologies. Here we describe an efficient single-strand ligation (90% of the input mRNAs) using a fluorescein-conjugated polyethylene glycol puromycin (Fluor-PEG Puro) spacer. This ligation provides a stable c-jun mRNA with a flexible Fluor-PEG Puro spacer for efficient fusion formation (70% of the input mRNA with the PEG spacer) in a cell-free wheat germ translation system. When using a 5' untranslated region including SP6 promoter and Omega29 enhancer (a part of tobacco mosaic virus Omega), an A(8) sequence (eight consecutive adenylate residues) at the 3' end is suitable for fusion formation, while an XA(8) sequence (XhoI and the A(8) sequence) is suitable for C-terminal protein labeling. Further, we report that Fluor-PEG N-t-butyloxycarbonylpuromycin [Puro(Boc)] spacer enhances the stability and efficiency of c-jun mRNA template for C-terminal protein labeling. These mRNA templates should be useful for puromycin-based technologies (fusion formation and C-terminal protein labeling) to facilitate high-throughput in vitro protein selection for not only evolutionary protein engineering, but also proteome exploration.

摘要

对于使用基因型（mRNA）-表型（蛋白质）融合形成和C末端蛋白质标记作为筛选后分析的高通量体外蛋白质筛选而言，提高这两种技术的mRNA模板的稳定性和效率非常重要。在此，我们描述了一种使用荧光素共轭聚乙二醇嘌呤霉素（Fluor-PEG Puro）间隔物的高效单链连接方法（90%的输入mRNA）。这种连接在无细胞小麦胚翻译系统中提供了一种带有灵活的Fluor-PEG Puro间隔物的稳定的c-jun mRNA，用于高效融合形成（70%带有PEG间隔物的输入mRNA）。当使用包含SP6启动子和Omega29增强子（烟草花叶病毒Omega的一部分）的5'非翻译区时，3'端的A(8)序列（八个连续的腺苷酸残基）适合融合形成，而XA(8)序列（XhoI和A(8)序列）适合C末端蛋白质标记。此外，我们报告Fluor-PEG N-叔丁氧羰基嘌呤霉素[Puro(Boc)]间隔物增强了用于C末端蛋白质标记的c-jun mRNA模板的稳定性和效率。这些mRNA模板对于基于嘌呤霉素的技术（融合形成和C末端蛋白质标记）应该是有用的，以便促进高通量体外蛋白质筛选，不仅用于进化蛋白质工程，也用于蛋白质组探索。