Graduate School of Engineering and Immunology Frontier Research Center, Osaka University , Suita, Osaka 565-0871, Japan.
Acc Chem Res. 2014 Jan 21;47(1):247-56. doi: 10.1021/ar400135f. Epub 2013 Aug 8.
The use of genetic engineering techniques allows researchers to combine functional proteins with fluorescent proteins (FPs) to produce fusion proteins that can be visualized in living cells, tissues, and animals. However, several limitations of FPs, such as slow maturation kinetics or issues with photostability under laser illumination, have led researchers to examine new technologies beyond FP-based imaging. Recently, new protein-labeling technologies using protein/peptide tags and tag-specific probes have attracted increasing attention. Although several protein-labeling systems are com mercially available, researchers continue to work on addressing some of the limitations of this technology. To reduce the level of background fluorescence from unlabeled probes, researchers have pursued fluorogenic labeling, in which the labeling probes do not fluoresce until the target proteins are labeled. In this Account, we review two different fluorogenic protein-labeling systems that we have recently developed. First we give a brief history of protein labeling technologies and describe the challenges involved in protein labeling. In the second section, we discuss a fluorogenic labeling system based on a noncatalytic mutant of β-lactamase, which forms specific covalent bonds with β-lactam antibiotics such as ampicillin or cephalosporin. Based on fluorescence (or Förster) resonance energy transfer and other physicochemical principles, we have developed several types of fluorogenic labeling probes. To extend the utility of this labeling system, we took advantage of a hydrophobic β-lactam prodrug structure to achieve intracellular protein labeling. We also describe a small protein tag, photoactive yellow protein (PYP)-tag, and its probes. By utilizing a quenching mechanism based on close intramolecular contact, we incorporated a turn-on switch into the probes for fluorogenic protein labeling. One of these probes allowed us to rapidly image a protein while avoiding washout. In the future, we expect that protein-labeling systems with finely designed probes will lead to novel methodologies that allow researchers to image biomolecules and to perturb protein functions.
基因工程技术的应用使得研究人员能够将功能蛋白与荧光蛋白（FPs）结合，产生融合蛋白，从而可以在活细胞、组织和动物中进行可视化。然而，FPs 的几个局限性，如成熟动力学缓慢或在激光照射下的光稳定性问题，促使研究人员研究超越 FP 成像的新技术。最近，使用蛋白质/肽标签和标签特异性探针的新蛋白质标记技术引起了越来越多的关注。尽管有几种蛋白质标记系统是商业上可用的，但研究人员仍在继续努力解决该技术的一些局限性。为了降低未标记探针的背景荧光水平，研究人员采用了荧光标记，其中标记探针在标记靶蛋白之前不会发出荧光。在本报告中，我们回顾了我们最近开发的两种不同的荧光蛋白质标记系统。首先，我们简要介绍了蛋白质标记技术的历史，并描述了蛋白质标记所涉及的挑战。在第二节中，我们讨论了基于非催化突变体β-内酰胺酶的荧光标记系统，该酶与氨苄青霉素或头孢菌素等β-内酰胺抗生素形成特异性共价键。基于荧光（或Förster）共振能量转移和其他物理化学原理，我们开发了几种类型的荧光标记探针。为了扩展该标记系统的实用性，我们利用疏水性β-内酰胺前药结构实现了细胞内蛋白质标记。我们还描述了一种小的蛋白质标签，即光活性黄色蛋白（PYP）-标签及其探针。通过利用基于紧密分子内接触的猝灭机制，我们将开环开关整合到荧光蛋白质标记探针中。其中一个探针允许我们在避免冲洗的情况下快速对蛋白质进行成像。在未来，我们预计具有精细设计探针的蛋白质标记系统将带来新的方法，使研究人员能够对生物分子进行成像并干扰蛋白质功能。
