Truncated Variants of Gaussia Luciferase with Tyrosine Linker for Site-Specific Bioconjugate Applications
Truncated Variants of Gaussia Luciferase with Tyrosine Linker for Site-Specific Bioconjugate Applications
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DOI：
10.1038/srep26814
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发表时间：
2016-06-08
影响因子：
4.6
通讯作者：
Deo, Sapna K.
中科院分区：
文献类型：
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作者：
Hunt, Eric A.;Moutsiopoulou, Angeliki;Deo, Sapna K.
Gaussia荧光素酶（Gluc）具有体积小、发光亮、稳定性好等优点，已成为生物发光检测领域的重要报道蛋白。然而，对功能结构形成至关重要的10个内部半胱氨酸残基使得在细菌系统中表达大量可溶性蛋白质变得困难。除了这一挑战之外，目前缺乏结构数据进一步使Gluc在体外应用中的使用复杂化，例如生物传感器或细胞递送，这两者都严重依赖于稳健和可重复的生物缀合技术。虽然Gluc对于荧光素酶来说已经相当小，但仍然保留显著生物发光活性的尺寸减小，结合化学修饰的更可再现的生物正交方法和细菌中的简易表达，将在生物传感器设计和细胞转运研究中非常有益。我们已经开发了截短的变体Gluc，保持有吸引力的生物发光功能，其特征在于它们的光谱和动力学特性。这些变体从细菌系统中大量纯化。此外，C-末端接头已被并入这些变体中，其可用于通过基于酪氨酸的生物缀合技术进行可靠的特异性修饰，从而使半胱氨酸残基的敏感网络不受干扰。
Gaussia luciferase (Gluc)-with its many favorable traits such as small size, bright emission, and exceptional stability-has become a prominent reporter protein for a wide range of bioluminescence-based detection applications. The ten internal cysteine residues crucial to functional structure formation, however, make expression of high quantities of soluble protein in bacterial systems difficult. In addition to this challenge, the current lack of structural data further complicates the use of Gluc for in vitro applications, such as biosensors, or cellular delivery, both of which rely heavily on robust and reproducible bioconjugation techniques. While Gluc is already appreciably small for a luciferase, a reduction in size that still retains significant bioluminescent activity, in conjunction with a more reproducible bioorthogonal method of chemical modification and facile expression in bacteria, would be very beneficial in biosensor design and cellular transport studies. We have developed truncated variants of Gluc, which maintain attractive bioluminescent features, and have characterized their spectral and kinetic properties. These variants were purified in high quantities from a bacterial system. Additionally, a C-terminal linker has been incorporated into these variants that can be used for reliable, specific modification through tyrosine-based bioconjugation techniques, which leave the sensitive network of cysteine residues undisturbed.