کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
1932886 | 1050596 | 2009 | 6 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: Multiply mutated Gaussia luciferases provide prolonged and intense bioluminescence Multiply mutated Gaussia luciferases provide prolonged and intense bioluminescence](/preview/png/1932886.png)
Gaussia luciferase (GLuc) from the copepod Gaussia 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.
Journal: Biochemical and Biophysical Research Communications - Volume 389, Issue 4, 27 November 2009, Pages 563–568