Hobson John P, Liu Shihui, Leppla Stephen H, Bugge Thomas H
Meso Scale Diagnostics, Gaithersburg, MD, USA.
Methods Mol Biol. 2009;539:115-29. doi: 10.1007/978-1-60327-003-8_7.
The scarcity of methods to visualize the activity of individual cell surface proteases in situ has hampered basic research and drug development efforts. In this chapter, we describe a simple, sensitive, and noninvasive assay that uses nontoxic reengineered bacterial cytotoxins with altered protease cleavage specificity to visualize specific cell surface proteolytic activity in single living cells. The assay takes advantage of the absolute requirement for site-specific endoproteolytic cleavage of cell surface-bound anthrax toxin protective antigen for its capacity to translocate an anthrax toxin lethal factor-beta-lactamase fusion protein to the cytoplasm. A fluorogenic beta-lactamase substrate is then used to visualize the cytoplasmically translocated anthrax toxin lethal factor-beta-lactamase fusion protein. By using anthrax toxin protective antigen variants that are reengineered to be cleaved by furin, urokinase plasminogen activator, or metalloproteinases, the cell surface activities of each of these proteases can be specifically and quantitatively determined with single cell resolution. The imaging assay is excellently suited for fluorescence microscope, fluorescence plate reader, and flow cytometry formats, and it can be used for a variety of purposes.
用于原位可视化单个细胞表面蛋白酶活性的方法稀缺，这阻碍了基础研究和药物开发工作。在本章中，我们描述了一种简单、灵敏且非侵入性的检测方法，该方法使用具有改变的蛋白酶切割特异性的无毒工程化细菌细胞毒素，以可视化单个活细胞中的特定细胞表面蛋白水解活性。该检测方法利用了细胞表面结合的炭疽毒素保护性抗原进行位点特异性内切蛋白水解切割对于其将炭疽毒素致死因子 - β - 内酰胺酶融合蛋白转运至细胞质的能力的绝对要求。然后使用一种荧光β - 内酰胺酶底物来可视化细胞质中转运的炭疽毒素致死因子 - β - 内酰胺酶融合蛋白。通过使用经工程改造可被弗林蛋白酶、尿激酶型纤溶酶原激活剂或金属蛋白酶切割炭疽毒素保护性抗原变体，这些蛋白酶各自的细胞表面活性可以通过单细胞分辨率进行特异性和定量测定。该成像检测方法非常适合荧光显微镜、荧光酶标仪和流式细胞仪检测形式，并且可用于多种目的。
