A new reporter gene system suited for cell-free protein synthesis and high-throughput screening in small reaction volumes.
The properties of M-hirudin as a new reporter gene system were examined using rabbit reticulocyte lysate for cell-free protein expression. In contrast to the luciferase gene, in vitro translation of M-hirudin is highly robust against changes in concentrations of K+ (and Rb+). In addition, M-hirudin can be detected very sensitively using a reasonably priced fluorimetric thrombin assay. To show that the new reporter gene system is well suited for (u)HTS-applications, cell-free synthesis as well as the fluorimetric assay of M-hirudin were carried out in nanotiter and microtiter plates, respectively.
Hempel
R
R
Abteilung fuer Molekulare Genetik und Praeparative Molekularbiologie, Institut fuer Mikrobiologie und Genetik, Grisebachstrasse 8, Goettingen, 37077, Germany.
Wirsching
F
F
Schober
A
A
Schwienhorst
A
A
D000818:Animals; D000998:Antiviral Agents / Q000032:analysis; D002474:Cell-Free System / Q000378:metabolism; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005470:Fluorometry / Q000379:methods; D017930:Genes, Reporter / Q000235:genetics*; D006629:Hirudins / Q000235:genetics* / Q000378:metabolism* / Q000494:pharmacology; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D011188:Potassium / Q000378:metabolism*; D014176:Protein Biosynthesis / Q000235:genetics / Q000502:physiology; D016372:RNA, Antisense / Q000032:analysis*; D012367:RNA, Viral / Q000032:analysis; D011817:Rabbits; D012413:Rubidium / Q000378:metabolism; D013917:Thrombin / Q000032:analysis* / Q000037:antagonists & inhibitors
D000998:Antiviral Agents; D006629:Hirudins; D016372:RNA, Antisense; D012367:RNA, Viral; D008156:Luciferases; D013917:Thrombin; D012413:Rubidium; D011188:Potassium