如何制造用于生物测定的功能性人工荧光素酶。

How to Fabricate Functional Artificial Luciferases for Bioassays.

作者信息

Kim Sung-Bae, Fujii Rika

机构信息

Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba, Ibaraki, 305-8569, Japan.

出版信息

Methods Mol Biol. 2016;1461:43-53. doi: 10.1007/978-1-4939-3813-1_4.

DOI:10.1007/978-1-4939-3813-1_4

PMID:27424894

Abstract

The present protocol introduces fabrication of artificial luciferases (ALuc(®)) by extracting the consensus amino acids from the alignment of copepod luciferase sequences. The made ALucs have unique sequential identities that are phylogenetically distinctive from those of any existing copepod luciferase. Some ALucs exhibited heat stability, and strong and greatly prolonged optical intensities. The made ALucs are applicable to various bioassays as an optical readout, including live cell imaging, single-chain probes, and bioluminescent tags of antibodies. The present protocol guides on how to fabricate a unique artificial luciferase with designed optical properties and functionalities.

摘要

本方案介绍了通过从桡足类荧光素酶序列比对中提取共有氨基酸来制备人工荧光素酶（ALuc(®)）的方法。所制备的ALuc具有独特的序列特征，在系统发育上与任何现有的桡足类荧光素酶都不同。一些ALuc表现出热稳定性，以及强烈且大大延长的光强度。所制备的ALuc可作为光学读数应用于各种生物测定，包括活细胞成像、单链探针和抗体的生物发光标签。本方案指导如何制备具有设计光学性质和功能的独特人工荧光素酶。