Suppr超能文献

鉴定单体红色荧光蛋白中可耐受肽插入的位点,并测试相应的环形排列。

Identification of sites within a monomeric red fluorescent protein that tolerate peptide insertion and testing of corresponding circular permutations.

作者信息

Li Yankun, Sierra Aillette M, Ai Hui-Wang, Campbell Robert E

机构信息

Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada.

出版信息

Photochem Photobiol. 2008 Jan-Feb;84(1):111-9. doi: 10.1111/j.1751-1097.2007.00206.x.

Abstract

In recent years the class of known fluorescent proteins (FPs) has dramatically expanded as an ever-increasing numbers of variants and homologs of the green fluorescent protein (GFP) from Aequorea jellyfish have been either engineered in the lab or discovered in other marine organisms. The red fluorescent protein (RFP) from Discosoma coral (also known as dsFP583 and DsRed) has proven to be a particularly fruitful progenitor of variants with biochemical and spectroscopic properties conducive to applications in live cell imaging. We have investigated the tolerance of an engineered monomeric descendent of Discosoma RFP, known as mCherry, towards peptide insertion and circular permutation. Starting from a random library of insertion variants, we identified six genetically distinct sites localized in three different loops where a sequence of five residues could be inserted without abolishing the ability of the protein to form its intrinsic red fluorescent chromophore. For each of these insertion variants, a corresponding circular permutation variant was created in which the original N- and C-termini were connected by a six-residue linker and new termini were introduced at the site of the insertion. All six circular permutation variants had significantly diminished brightness relative to the analogous insertion variants. The most promising circular permutation variant has termini at the position corresponding to residue 184 of mCherry and retains 37% of the intrinsic fluorescent brightness of mCherry. These circularly permuted variants may serve as the foundation for construction of genetically encoded Ca2+ sensors analogous to the previously reported camgaroo, pericam and G-CaMP sensors based on variants of Aequorea GFP.

摘要

近年来,随着越来越多源自维多利亚水母绿色荧光蛋白(GFP)的变体和同源物在实验室中被改造出来或在其他海洋生物中被发现,已知荧光蛋白(FP)的种类急剧增加。来自盘状珊瑚的红色荧光蛋白(RFP,也称为dsFP583和DsRed)已被证明是变体的一个特别丰富的来源,其生化和光谱特性有利于在活细胞成像中应用。我们研究了盘状珊瑚RFP的一种工程改造的单体后代mCherry对肽插入和环形排列的耐受性。从插入变体的随机文库开始,我们确定了位于三个不同环中的六个遗传上不同的位点,在这些位点可以插入五个残基的序列而不消除蛋白质形成其固有红色荧光发色团的能力。对于这些插入变体中的每一个,都创建了一个相应的环形排列变体,其中原始的N端和C端通过一个六残基的接头连接,并且在插入位点引入了新的末端。所有六个环形排列变体相对于类似的插入变体亮度都显著降低。最有前途的环形排列变体在与mCherry的第184位残基对应的位置有末端,并且保留了mCherry固有荧光亮度的37%。这些环形排列变体可以作为构建基因编码的Ca2+传感器的基础,类似于先前报道的基于维多利亚水母GFP变体的camgaroo、pericam和G-CaMP传感器。

文献AI研究员

20分钟写一篇综述,助力文献阅读效率提升50倍。

立即体验

用中文搜PubMed

大模型驱动的PubMed中文搜索引擎

马上搜索

文档翻译

学术文献翻译模型,支持多种主流文档格式。

立即体验