Issues luciferase

Following this mechanism, luciferase-bound reduced FMN (FMNH intermediate I) reacts with oxygen to form the 4a-hydroperoxy-4a,5-dihydroFMN intermediate II (HF-OCT). The addition of a long-chain aliphatic aldehyde generates the 4a-peroxyhemiacetal-4a,5-dihydroFMN intermediate III (HF-OOCH(OH)R), which is subsequently converted to a radical pair of 4a-hydroxy-4a,5-dihydroFMN radical cation (IV+ HF-OH+ ) and a carboxylic acid radical anion RC(0H)0" A 1-e transfer from RC(0H)0- to IV+ produces the excited 4a-hydoxy-4a,5-dihydroFMN intermediate IV. Relaxation of IV to the ground state produces bio luminescence ( max 490 nm) with a quantum yield of about 0.16. Finally, IV decays to form FMN and water. The present report addresses several key issues of this mechanism with respect to the identity of the proposed excited emitter HF-OH, the energetics of its formation, and the requirement of a hydrophobic luciferase active site for a high quantum yield of the emitter. 

For the specific use of measuring transcriptional activity, the method provides several unique advantages however, several issues must be treated with great care and simple quantification of light emission may not always provide a true representation of the biological effect studied. First, as luciferase expression is... 

It is important to be able to readily produce sufficient amounts of recombinant Luciferase-fused protein(s) in order to perform high-throughput screening. We have found that recombinant protein production by in vitro-transcription/translation (IVT), bacterial expression, or the /baculovirus systems all work well. Protein production by IVT is the quickest and easiest of the three, although the limited protein yield can be an issue. A much greater protein yield can be obtained using the bacterial or /baculovirus systems, but these are much more labor intensive. 

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