Towards Water-Free Biobanks: Long-Term Dry-Preservation At Room Temperature Of Desiccation-Sensitive Enzyme Luciferase In Air-Dried Insect Cells
Towards Water-Free Biobanks: Long-Term Dry-Preservation At Room Temperature Of Desiccation-Sensitive Enzyme Luciferase In Air-Dried Insect Cells
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迈向无水生物库：风干昆虫细胞中干燥敏感酶荧光素酶在室温下长期干燥保存
DOI：
10.1101/120063
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发表时间：
2017
期刊：
影响因子：
--
通讯作者：
Takahiro Kikawada
中科院分区：
文献类型：
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作者：
Shingo Kikuta;Shunsuke J Watanabe;Ryoichi Sato;Oleg Gusev;Alexander Nesmelov;Yoichiro Sogame;Richard Cornette;Takahiro Kikawada
来自脱水性吸浆虫胚胎的耐脱水培养细胞Pv 11在无代谢状态下耐受完全脱水，并在再水化后恢复其代谢。这些特征使我们开发了一种新的酶干燥保存技术，因为目前还不清楚Pv 11细胞是否可以在干燥状态下保存外源酶。这项研究表明，Pv 11细胞保护外源干燥敏感酶，荧光素酶（Luc），即使在室温下干燥储存372天后仍保持酶活性。包括与海藻糖预孵育、脱水、储存和再水化的过程允许稳定表达荧光素酶的Pv 11（Pv 11-Luc）细胞在干燥后存活，并且在再水化后仍然发射由荧光素酶引起的发光。荧光素酶在Pv 11-Luc细胞再水化后产生的发光在翻译抑制剂的存在下没有显著降低，表明活性并不来源于细胞代谢恢复后的novoenzyme合成。这些发现表明，存活的Pv 11细胞在干燥过程中几乎完全保护荧光素酶。缺乏预孵育步骤导致再水化后荧光素酶活性丧失。我们发现，与Pv 11细胞中脱水耐受性相关基因诱导相关的海藻糖预孵育允许有效地在干燥状态下的酶的活体保存。
Desiccation-tolerant cultured cells Pv11 derived from the anhydrobiotic midge embryo endure complete desiccation in an ametabolic state and resume their metabolism after rehydration. These features led us to develop a novel dry preservation technology for enzymes as it was still unclear whether Pv11 cells could preserve an exogenous enzyme in the dry state. This study shows that Pv11 cells protect an exogenous desiccation-sensitive enzyme, luciferase (Luc), preserving the enzymatic activity even after dry storage for 372 days at room temperature. A process including preincubation with trehalose, dehydration, storage, and rehydration allowed Pv11 (Pv11-Luc) cells stably expressing luciferase to survive desiccation and still emit luminescence caused by luciferase after rehydration. Luminescence produced by luciferase in Pv11-Luc cells after rehydration did not significantly decrease in presence of a translation inhibitor, showing that the activity did not derive fromde novoenzyme synthesis following the resumption of cell metabolism. These findings indicate that the surviving Pv11 cells almost completely protect luciferase during desiccation. Lacking of the preincubation step resulted in the loss of luciferase activity after rehydration. We showed that preincubation with trehalose associated to induction of desiccation tolerance-related genes in Pv11 cells allowed effectivein vivopreservation of enzymes in the dry state.