Engineering and Characterization of 3-Aminotyrosine-Derived Red Fluorescent Variants of Circularly Permutated Green Fluorescent Protein.
Engineering and Characterization of 3-Aminotyrosine-Derived Red Fluorescent Variants of Circularly Permutated Green Fluorescent Protein.
复制标题
DOI：
10.3390/bios14010054
复制
发表时间：
2024-01-20
期刊：
影响因子：
--
通讯作者：
中科院分区：
文献类型：
--
作者：
将非正则氨基酸3-氨基酪氨酸(AY)引入绿色荧光蛋白(GFP)类发色团，有望实现红移荧光。然而，不一致的结果，包括不想要的绿色荧光物种，阻碍了这一方法的有效性。在本研究中，我们优化了Ay衍生的cpGFP(Ay-cpGFP)的表达条件。丰富的培养基和诱导前后的限氧等关键因素使红移蛋白的高产、高纯度生产成为可能。我们还设计了两个变种的ay-cpGFP，通过突变发色团周围的几个氨基酸残基来增强亮度。我们进一步研究了AY衍生蛋白对金属离子、活性氧物种(ROS)和活性氮物种(RNS)的敏感性。在cpGFP中加入Ay对金属离子的反应性影响很小，但增加了对RNS的响应。在这些发现的基础上，我们检测了ay-cpGFP在哺乳动物细胞中的表达，发现培养液中的还原剂显著增加了红色发光产物。我们的研究表明，优化表达条件以促进细胞状态的降低被证明在大肠杆菌和哺乳动物细胞中都能有效地产生所需的红色发光产物，而基于靶向突变的蛋白质工程可以进一步增强亮度和方法的稳健性。
Introducing 3-aminotyrosine (aY), a noncanonical amino acid (ncAA), into green fluorescent protein (GFP)-like chromophores shows promise for achieving red-shifted fluorescence. However, inconsistent results, including undesired green fluorescent species, hinder the effectiveness of this approach. In this study, we optimized expression conditions for an aY-derived cpGFP (aY-cpGFP). Key factors like rich culture media and oxygen restriction pre- and post-induction enabled high-yield, high-purity production of the red-shifted protein. We also engineered two variants of aY-cpGFP with enhanced brightness by mutating a few amino acid residues surrounding the chromophore. We further investigated the sensitivity of the aY-derived protein to metal ions, reactive oxygen species (ROS), and reactive nitrogen species (RNS). Incorporating aY into cpGFP had minimal impact on metal ion reactivity but increased the response to RNS. Expanding on these findings, we examined aY-cpGFP expression in mammalian cells and found that reductants in the culture media significantly increased the red-emitting product. Our study indicates that optimizing expression conditions to promote a reduced cellular state proved effective in producing the desired red-emitting product in both E. coli and mammalian cells, while targeted mutagenesis-based protein engineering can further enhance brightness and increase method robustness.
登录
查看更多内容
影响因子：
4.6
作者：
Saha R;Verma PK;Rakshit S;Saha S;Mayor S;Pal SK
通讯作者：
Pal SK
影响因子：
4.8
作者：
Corker, H;Poole, RK
通讯作者：
Poole, RK
影响因子：
3.8
作者：
Cesme, Mustafa
通讯作者：
Cesme, Mustafa
影响因子：
3
作者：
Carballal, Sebastian;Bartesaghi, Silvina;Radi, Rafael
通讯作者：
Radi, Rafael
影响因子：
16.6
作者：
Ermakova YG;Bilan DS;Matlashov ME;Mishina NM;Markvicheva KN;Subach OM;Subach FV;Bogeski I;Hoth M;Enikolopov G;Belousov VV
通讯作者：
Belousov VV