Fluorescent Protein Variants Generated by Reassembly between Skeleton and Chromophore.
Fluorescent Protein Variants Generated by Reassembly between Skeleton and Chromophore.
复制标题
骨架和发色团之间重新组装产生的荧光蛋白变体。
DOI：
10.1021/acsomega.0c05299
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发表时间：
2021-02-02
期刊：
影响因子：
4.1
通讯作者：
Gao X
中科院分区：
文献类型：
--
作者：
Sun T;Li T;Yi K;Yan G;Gao X
荧光蛋白（FPs）可以作为内在分子标签来跟踪活细胞的动态活性。以一种可用的和大量的方式获得变体一直是一个挑战。在这里，我们采用基于计算机的微阵列合成方法来实现发色团与骨架之间的重组。使用DNAWorks将输入的FP模板分割成一组重叠的寡核苷酸（20-43 mer），退火温度、G + C含量和密码子频率平衡。通过在分割过程中切换分裂位点，使发色团的结构保持在同一截面上，并对密码子进行优化，进一步保持参数的平衡。设计的寡核苷酸在光可编程微流控阵列上合成。序列分析和随后的条件诱导表达表明，寡核苷酸被高度重组。光谱、光稳定性和随机选择变异的分子大小检测表明，它们是不同的单体蛋白，保留了光活性。我们的研究提供了一种基于计算机设计的并行合成获得FP变体的有效方法。
Fluorescent proteins (FPs) can be used as intrinsic molecular tags to track the dynamic activity in live cells. To obtain variants in an available and massive manner is always a challenge. Here, we adopted a computer-based microarray synthesis method to realize the reassembly between the chromophore and the skeleton. DNAWorks was used to segment the input FP templates into a set of overlapping oligonucleotides (20–43 mer) with a balanced annealing temperature, G + C content, and codon frequency. The constitution of the chromophore was kept in the same section by switching the divided sites during segmentation and the codon was optimized to further keep the balanced parameters. The designed oligonucleotides were synthesized on photo-programmable microfluidic arrays. Sequence analysis and the subsequent conditional induced expression of FPs revealed that oligonucleotides were highly reassembled. Spectra, photostability, and molecular size detection of randomly selected variants showed that they were distinct monomeric proteins that preserved photoactivity. Our study provides an effective means of obtaining FP variants based on a computer-designed parallel synthesis.
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