Chiang Jason Jui-Hsuan, Li Isaac, Truong Kevin
Institute of Biomaterials and Biomedical Engineering, University of Toronto, 4 Taddle Creek Road, M5S 3G9, Toronto, Ont., Canada.
Biotechnol Lett. 2006 Apr;28(7):471-5. doi: 10.1007/s10529-006-0007-6.
By experimenting with many different circularly permutated yellow fluorescent protein (cpYFP) variants as acceptors in fluorescence resonance energy transfer based biosensors, the optimal dynamic range can be discovered by sampling the possibilities of relative fluorophore orientations before and after bioactivity. Hence, to facilitate the sampling process, we introduced a new approach to construct a library of cpYFP variants using fluorescence screening and a tandem fusion template. This new approach is rapid because it does not require creating intermediate N- and C-terminal fragments and it allows quick screening for positive colonies by fluorescence. As a demonstration, eleven cpYFP variants were created and eight showed fluorescence. The emission and excitation spectra of these cpYFP variants showed strong similarity to YFP and therefore can be used in replacement.
通过在基于荧光共振能量转移的生物传感器中试验许多不同的环状排列黄色荧光蛋白（cpYFP）变体作为受体，可以通过对生物活性前后相对荧光团取向的可能性进行采样来发现最佳动态范围。因此，为了促进采样过程，我们引入了一种新方法，利用荧光筛选和串联融合模板构建cpYFP变体文库。这种新方法速度很快，因为它不需要创建中间的N端和C端片段，并且可以通过荧光快速筛选阳性菌落。作为一个示范，创建了11个cpYFP变体，其中8个显示出荧光。这些cpYFP变体的发射光谱和激发光谱与黄色荧光蛋白（YFP）非常相似，因此可以用来替代YFP。
