RT Journal Article
SR Electronic
T1 Towards water-free biobanks: long-term dry-preservation at room temperature of desiccation-sensitive enzyme luciferase in air-dried insect cells
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 120063
DO 10.1101/120063
A1 Kikuta, Shingo
A1 Watanabe, Shunsuke J.
A1 Sato, Ryoichi
A1 Gusev, Oleg
A1 Nesmelov, Alexander
A1 Sogame, Yoichiro
A1 Cornette, Richard
A1 Kikawada, Takahiro
YR 2017
UL http://biorxiv.org/content/early/2017/03/24/120063.abstract
AB Desiccation-tolerant cultured cells Pv11 derived from the anhydrobiotic Polypedilum vanderplanki embryo endure complete desiccation because of their 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 preserved an exogenous enzyme in the dry state. This study shows that Pv11 cells protect an exogenous desiccation-sensitive enzyme, luciferase, preserving the enzymatic activity even after dry storage for 372 days at room temperature. A process including pre-incubation with trehalose, dehydration, storage, and rehydration allowed Pv11 (Pvll-Luc) cells stably expressing luciferase to survive desiccation and still emit luminescence caused by luciferase after rehydration. Luminescence produced by luciferase in Pvll-Luc cells after rehydration did not significantly decrease in presence of a translation inhibitor, showing that the activity did not derive from de novo enzyme 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-tolerant related genes in Pv11 cells allowed effective in vivo preservation of enzymes in the dry state.LucluciferaseGFPgreen fluorescent proteinIPTGisopropyl-β-D-thiogalactopyranosideLEAlate embryogenesis abundantHEK293Thuman embryonic kidney 293TBSAbovine serum albuminFBSfetal bovine serumD-MEMDulbecco's modified Eagle mediumERendoplasmic reticulumRHrelative humidity