Conditional protein splicing: A new tool to control protein structure and function in vitro and in vivo
Conditional protein splicing: A new tool to control protein structure and function in vitro and in vivo
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DOI：
10.1021/ja0362813
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发表时间：
2003-09-03
影响因子：
15
通讯作者：
Muir, TW
中科院分区：
文献类型：
--
作者：
Mootz, HD;Blum, ES;Muir, TW
蛋白质剪接是一种自然发生的过程，其中插入内含肽结构域以自催化方式将其自身从前体多肽中切除，同时通过天然肽键连接两个侧翼外显肽序列。我们最近报道了一种工程化的分裂VMA内含肽，其两个多肽之间的反式剪接活性可以由小分子雷帕霉素触发。在这份报告中，我们证明了这种条件蛋白剪接（CPS）系统可用于哺乳动物细胞。瞬时转染后，两个含有麦芽糖结合蛋白 (MBP) 和 His 标签作为外显肽的模型构建体从组成型启动子表达。在用雷帕霉素或其无毒类似物处理的细胞中通过蛋白质印迹和免疫沉淀检测剪接产物MBP-His。在没有小分子诱导物的情况下，24 小时内没有观察到背景剪接。添加雷帕霉素后 10 分钟内即可检测到产物形成，这表明 CPS 的翻译后性质具有快速反应的优势。蛋白质剪接水平呈剂量依赖性，并且可以与小分子子囊霉素竞争性减弱。在相关研究中，利用一系列纯化蛋白质研究了 CPS 成分的几何灵活性。 FKBP 和 FRB 结构域被雷帕霉素二聚化，从而诱导分裂内含肽的重建，并与分裂内含肽半部分的外显肽序列融合。当FKBP和FRB占据一个或两个外显肽位置时，雷帕霉素仍然会触发CPS。这一发现表明 CPS 在蛋白质组学领域的进一步应用。总之，CPS 有望成为一种强大的新工具，在体外和活细胞中控制蛋白质结构和功能。
Protein splicing is a naturally occurring process in which an intervening intein domain excises itself out of a precursor polypeptide in an autocatalytic fashion with concomitant linkage of the two flanking extein sequences by a native peptide bond. We have recently reported an engineered split VMA intein whose splicing activity in trans between two polypeptides can be triggered by the small molecule rapamycin. In this report, we show that this conditional protein splicing (CPS) system can be used in mammalian cells. Two model constructs harboring maltose-binding protein (MBP) and a His-tag as exteins were expressed from a constitutive promoter after transient transfection. The splicing product MBP-His was detected by Western blotting and immunoprecipitation in cells treated with rapamycin or a nontoxic analogue thereof. No background splicing in the absence of the small-molecule inducer was observed over a 24-h time course. Product formation could be detected within 10 min of addition of rapamycin, indicating the advantage of the posttranslational nature of CPS for quick responses. The level of protein splicing was dose dependent and could be competitively attenuated with the small molecule ascomycin. In related studies, the geometric flexibility of the CPS components was investigated with a series of purified proteins. The FKBP and FRB domains, which are dimerized by rapamycin and thereby induce the reconstitution of the split intein, were fused to the extein sequences of the split intein halves. CPS was still triggered by rapamycin when FKBP and FRB occupied one or both of the extein positions. This finding suggests yet further applications of CPS in the area of proteomics. In summary, CPS holds great promise to become a powerful new too] to control protein structure and function in vitro and in living cells.