Genetically Encoded Fluorescent Biosensor for Rapid Detection of Protein Expression,ACS Synthetic Biology

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Genetically Encoded Fluorescent Biosensor for Rapid Detection of Protein Expression
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2020-10-12 , DOI: 10.1021/acssynbio.0c00407
Matthew G Eason ₁ , Antonia T Pandelieva ₁ , Marc M Mayer ₁ , Safwat T Khan ₁ , Hernan G Garcia _{2,

3,

4,

5} , Roberto A Chica ₁

Affiliation

Fluorescent proteins are widely used as fusion tags to detect protein expression in vivo. 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 in vivo following the specific and rapid binding of a protein tag (K_d 120 nM, k_on 1.7 × 10⁵ M^–1 s^–1). In live E. coli 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.

中文翻译：

用于快速检测蛋白质表达的基因编码荧光生物传感器

荧光蛋白被广泛用作融合标签来检测体内蛋白质的表达。为了发出荧光，这些蛋白质必须经历发色团成熟，这是一个缓慢的过程，半衰期为 5 到 >30 分钟，导致蛋白质表达实时检测的延迟。在这里，我们设计了一种基因编码的荧光生物传感器，能够在几秒钟内检测活细菌中的蛋白质表达。这种用于瞬时表达蛋白 (STEP) 的传感器基于完全成熟但暗淡的绿色荧光蛋白，在蛋白质标签特异性快速结合后，体内预先存在的荧光增加 11 倍（K _d 120 nM，k _on 1.7 × 10 ⁵ M^–1秒^–1 )。在活的大肠杆菌细胞中，我们的 STEP 生物传感器能够以比使用标准荧光蛋白融合物快两倍的速度检测蛋白质表达。我们的生物传感器为实时研究具有高时空分辨率的活细胞中的短时间尺度过程打开了大门。

更新日期：2020-11-21

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