Systems Bioinformatics/AIMMS, Vrije Universiteit Amsterdam, De Boelelaan 1085, 1081 HV, Amsterdam, The Netherlands.
Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH, Amsterdam, The Netherlands.
Sci Rep. 2019 Feb 19;9(1):2234. doi: 10.1038/s41598-019-38913-z.
Fluorescent proteins (FPs) are widely used in many organisms, but are commonly characterised in vitro. However, the in vitro properties may poorly reflect in vivo performance. Therefore, we characterised 27 FPs in vivo using Saccharomyces cerevisiae as model organism. We linked the FPs via a T2A peptide to a control FP, producing equimolar expression of the 2 FPs from 1 plasmid. Using this strategy, we characterised the FPs for brightness, photostability, photochromicity and pH-sensitivity, achieving a comprehensive in vivo characterisation. Many FPs showed different in vivo properties compared to existing in vitro data. Additionally, various FPs were photochromic, which affects readouts due to complex bleaching kinetics. Finally, we codon optimized the best performing FPs for optimal expression in yeast, and found that codon-optimization alters FP characteristics. These FPs improve experimental signal readout, opening new experimental possibilities. Our results may guide future studies in yeast that employ fluorescent proteins.
荧光蛋白（FPs）广泛应用于许多生物中，但通常在体外进行特征描述。然而，体外特性可能无法很好地反映体内性能。因此，我们使用酿酒酵母作为模型生物，在体内对 27 种 FPs 进行了特征描述。我们通过 T2A 肽将 FPs 与对照 FP 连接起来，从 1 个质粒中以等摩尔的方式表达这 2 个 FP。通过这种策略，我们对 FPs 的亮度、光稳定性、光致变色性和 pH 敏感性进行了特征描述，实现了全面的体内特征描述。与现有的体外数据相比，许多 FPs 在体内表现出不同的特性。此外，各种 FPs 具有光致变色性，这会由于复杂的漂白动力学而影响读数。最后，我们对表现最好的 FPs 进行了密码子优化，以在酵母中获得最佳表达，并且发现密码子优化会改变 FP 特性。这些 FP 提高了实验信号的读出，开辟了新的实验可能性。我们的研究结果可能会指导未来在酵母中使用荧光蛋白的研究。
