Department of Chemistry and Biomolecular Sciences, University of Ottawa, 10 Marie Curie, Ottawa, Ontario K1N 6N5, Canada.
Biophysics Graduate Group, University of California, Berkeley, Berkeley, California 94720, United States.
ACS Synth Biol. 2020 Nov 20;9(11):2955-2963. doi: 10.1021/acssynbio.0c00407. Epub 2020 Oct 12.
Fluorescent proteins are widely used as fusion tags to detect protein expression . To become fluorescent, these proteins must undergo chromophore maturation, a slow process with a half-time of 5 to >30 min that causes delays in real-time detection of protein expression. Here, we engineer a genetically encoded fluorescent biosensor to enable detection of protein expression within seconds in live bacteria. This sensor for transiently expressed proteins (STEP) is based on a fully matured but dim green fluorescent protein in which pre-existing fluorescence increases 11-fold following the specific and rapid binding of a protein tag ( 120 nM, 1.7 × 10 M s). In live cells, our STEP biosensor enables detection of protein expression twice as fast as the use of standard fluorescent protein fusions. Our biosensor opens the door to the real-time study of short timescale processes in live cells with high spatiotemporal resolution.
荧光蛋白被广泛用作融合标签来检测蛋白质表达。为了变得具有荧光性，这些蛋白质必须经历发色团成熟过程，这是一个缓慢的过程，半衰期为 5 至> 30 分钟，导致实时检测蛋白质表达的延迟。在这里，我们设计了一种遗传编码的荧光生物传感器，能够在活细菌中在几秒钟内检测蛋白质表达。这种用于瞬时表达蛋白的传感器（STEP）基于完全成熟但暗淡的绿色荧光蛋白，在该蛋白中，在特定且快速结合蛋白标签后，预先存在的荧光增加了 11 倍（120 nM，1.7×10^7 M s^-1）。在活细胞中，我们的 STEP 生物传感器使蛋白表达的检测速度比使用标准荧光蛋白融合体快两倍。我们的生物传感器为实时研究活细胞中具有高时空分辨率的短时间尺度过程开辟了道路。
