Multiply mutated Gaussia luciferases provide prolonged and intense bioluminescence
Multiply mutated Gaussia luciferases provide prolonged and intense bioluminescence
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DOI：
10.1016/j.bbrc.2009.09.006
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发表时间：
2009-11-27
影响因子：
3.1
通讯作者：
Swartz, James R.
中科院分区：
文献类型：
--
作者：
Welsh, John P.;Patel, Kedar G.;Swartz, James R.
来自桡足类Caussia princeps的Gaussia luciferase（GLuc）是已知的最小和最亮的荧光素酶。GLuc催化腔肠素氧化产生强烈的蓝光，但发射半衰期很短。我们报告突变的葡萄糖具有更长的发光半衰期，保持相同的初始强度作为野生型酶。GLuc变体是使用无细胞蛋白质合成生产的，以提供高产量和快速生产完全活性的产品以及简单的非天然氨基酸取代。通过将homopropargylglycine和连接PEG使用叠氮化物-炔点击反应，我们还表明，在GLuc的四个蛋氨酸是表面可访问的。突变体为体内和体外研究提供了显著改善的报告蛋白，并且成功的非天然氨基酸掺入和PEG附着表明使用点击附着反应产生有用的生物缀合物的可行性。(C)2009爱思唯尔公司All rights reserved.
Gaussia luciferase (GLuc) from the copepod Caussia princeps is both the smallest and brightest known luciferase. GLuc catalyzes the oxidation of coelenterazine to produce an intense blue light but with a very short emission half-life. We report mutated GLucs with much longer luminescence half-lives that retain the same initial intensity as the wild-type enzyme. The GLuc variants were produced using cell-free protein synthesis to provide high yields and rapid production of fully active product as well as simple non-natural amino acid substitution. By incorporating homopropargylglycine and attaching PEG using azide-alkyne click reactions, we also show that the four methionines in GLuc are surface accessible. The mutants provide a significantly improved reporter protein for both in vivo and in vitro studies, and the Successful non-natural amino acid incorporation and PEG attachment indicate the feasibility of producing useful bioconjugates using click attachment reactions. (C) 2009 Elsevier Inc. All rights reserved.