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首页> 外文会议>12th Symposium on Bioluminescence and Chemiluminescence, Apr 5-9, 2002, Robinson College, University of Cambridge, UK >NOVEL IN VIVO REPORTERS BASED ON FIREFLY LUCIFERASE
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NOVEL IN VIVO REPORTERS BASED ON FIREFLY LUCIFERASE

机译：基于完全LUCIFERase的体内报道新颖

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摘要

Recombinant firefly luciferases offer the potential to create novel bioluminescent reporter enzymes with enhanced properties for in vivo imaging. Increased thermostability and changes in the spectrum of emitted light are two parameters that could improve detection sensitivity in situations where signal attenuation is problematic e.g. in vivo imaging of mammalian tissues. Recently, we have identified mutations in the North American firefly Photinus pyralis that affect both thermostability and wavelength of emitted light. A library containing all possible amino acid permutations at these positions has previously been constructed, expressed in Escherichia. coli, and screened for thermostability and changes to in vivo spectra. Here we describe the overexpression and purification of two mutant luciferases from this library. The in vitro properties of the purified enzymes are characterised and assessed for their suitability in both in vivo and in vitro assays.
机译：重组萤火虫荧光素酶具有创造新型生物发光报告酶的潜力，该酶具有增强的体内成像特性。增加的热稳定性和发射光光谱的变化是两个参数，可在信号衰减有问题的情况下（例如信号强度）提高检测灵敏度。哺乳动物组织的体内成像。最近，我们发现了北美萤火虫Photinus pyralis中的突变会影响热稳定性和发射光的波长。先前已经构建了在这些位置上包含所有可能的氨基酸排列的文库，并在大肠杆菌中表达。大肠杆菌，并筛选热稳定性和体内光谱的变化。在这里，我们描述了从该文库中两种突变荧光素酶的过表达和纯化。表征纯化的酶的体外特性，并评估其在体内和体外测定中的适用性。

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