Widdick David A, Eijlander Robyn T, van Dijl Jan Maarten, Kuipers Oscar P, Palmer Tracy
Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, UK.
J Mol Biol. 2008 Jan 18;375(3):595-603. doi: 10.1016/j.jmb.2007.11.002. Epub 2007 Nov 12.
We have developed a reporter protein system for the experimental verification of twin-arginine signal peptides. This reporter system is based on the Streptomyces coelicolor agarase protein, which is secreted into the growth medium by the twin-arginine translocation (Tat) pathway and whose extracellular activity can be assayed colorimetrically in a semiquantitative manner. Replacement of the native agarase signal peptide with previously characterized twin-arginine signal peptides from other Gram-positive and Gram-negative bacteria resulted in efficient Tat-dependent export of agarase. Candidate twin-arginine signal peptides from archaeal proteins as well as plant thylakoid-targeting sequences were also demonstrated to mediate agarase translocation. A naturally occurring variant signal peptide with an arginine-glutamine motif instead of the consensus di-arginine was additionally recognized as a Tat-targeting sequence by Streptomyces. Application of the agarase assay to previously uncharacterized candidate Tat signal peptides from Bacillus subtilis identified two further probable Tat substrates in this organism. This is the first versatile reporter system for Tat signal peptide identification.
我们开发了一种报告蛋白系统，用于对双精氨酸信号肽进行实验验证。该报告系统基于天蓝色链霉菌琼脂酶蛋白，该蛋白通过双精氨酸转运（Tat）途径分泌到生长培养基中，其细胞外活性可以通过比色法进行半定量测定。用先前鉴定的来自其他革兰氏阳性和革兰氏阴性细菌的双精氨酸信号肽替换天然琼脂酶信号肽，导致琼脂酶通过Tat途径有效输出。来自古细菌蛋白的候选双精氨酸信号肽以及植物类囊体靶向序列也被证明可介导琼脂酶转运。链霉菌还额外识别出一种天然存在的具有精氨酸 - 谷氨酰胺基序而非共有双精氨酸基序的变异信号肽作为Tat靶向序列。将琼脂酶测定应用于先前未表征的来自枯草芽孢杆菌的候选Tat信号肽，在该生物体中又鉴定出另外两种可能的Tat底物。这是第一个用于鉴定Tat信号肽的通用报告系统。
