Enhancing the thermal stability of a single-chain Fv fragment by in vivo global fluorination of the proline residues
Enhancing the thermal stability of a single-chain Fv fragment by in vivo global fluorination of the proline residues
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DOI：
10.1039/c0mb00154f
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发表时间：
2011-01-01
影响因子：
--
通讯作者：
Lee, Sun-Gu
中科院分区：
文献类型：
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作者：
Edwardraja, Selvakumar;Sriram, Sokalingam;Lee, Sun-Gu
单链Fv （scFv）格式蛋白是一种常用的诊断和治疗分析工具。scFv抗体片段在治疗应用中的使用表明，它们需要具有高热稳定性。过去人们描述了许多合理或不合理的方法，通过修饰天然氨基酸来设计或提高scFv蛋白的稳定性。在这里，我们已经证明了一种替代策略，以有效地提高非规范氨基酸的scFvs的热稳定性。以前，氟脯氨酸已被证明是一种调整许多多肽和少数球状蛋白稳定性的选择。因此，我们利用氟脯氨酸来取代scFv骨架区域上影响折叠或稳定性的天然脯氨酸，从而提高scFv的热稳定性。为了证明我们的方法，我们使用了一种细菌细胞质可折叠人源化抗c- met抗体（hu-MscFv）。利用细菌营养不良系统的体内残留特异性全局替代方法，成功地将hu-MscFv脯氨酸位点与(2S, 4R)-4-氟脯氨酸结合，而不影响其结构和功能。温度对氟化h_ - mscfv活性的时间依赖效应表明，与天然脯氨酸相比，氟化h_ - mscfv在40℃时稳定性提高，半衰期延长。氟脯氨酸对hu-MscFv的进一步基于模型的结构分析表明，氟原子能够在其局部微环境中与邻近的极性基团建立新的有利的偶极相互作用，从而使其改善的热稳定性合理化。此外，基于scFv序列的统计分析有力地支持了这一事实，即该方法可以应用于任何目标scFv，因为它们在其框架区域中含有高频保守的脯氨酸位点。
Single-chain Fv (scFv) format protein is a commonly used analytical tool for diagnostic and therapeutic applications. The usage of scFv antibody fragments in therapeutic applications has demonstrated that they need to have high thermostability. Many rational or irrational methods have been described erstwhile to engineer or improve the stability of scFv proteins by modifications of natural amino acid. Here we have demonstrated an alternate strategy to efficiently improve the thermostability of scFvs by non-canonical amino acid. Previously, fluoroprolines have been proven as a choice to tune the stability of many polypeptides and few globular proteins. Hence we exploited the usage of fluoroproline to enhance the thermal stability of scFv by replacing the natural proline on the framework regions of scFv that influence the folding or stability. To demonstrate our approach, a bacterial cytoplasmic foldable and humanized anti-c-Met scFv (hu-MscFv) was used. The hu-MscFv proline sites were successfully incorporated with (2S, 4R)-4-fluoroproline without affecting its structure and function by the in vivo residue specific global replacement method which exploits bacterial auxotrophic system. The time-dependent temperature effect on the activity of fluorinated hu-MscFv revealed its increased stability at 40 degrees C along with improved half-life than the hu-MscFv with natural proline. Further model based structure analysis on hu-MscFv with fluoroproline indicated that the fluorine atoms were able to establish new favourable dipole interactions with neighbouring polar groups in their local micro environments that rationalizes its improved thermostability. Moreover the scFv sequence based statistical analysis strongly supports the fact that this method can be applied to any target scFv, since they contain high frequency conserved proline sites in their framework regions.