On 2017 Mar 06, Misha Koksharov commented:

A really nice FP tool and screening/engineering work.

I have a few questions:

1) "Bioluminescence characterization in vitro. Each protein was diluted to 2 nM in 30mM MOPS 100mM KCl pH 7.5 with 0.1% BSA, and a final concentration of 20 μM substrate was used."

a) Do you mean you have used 20 μM for both LH2 and ATP in case of firefly luciferase (Ppy luc2)?

b) Was Mg²⁺ added to the reaction buffer in case of Fluc?

2) "Hexahistidine-tagged proteins were expressed in E. coli at 30°C for 30 h"

How active was Fluc after growing E.coli at 30°C? I'd expect that this should noticeably decrease its specific activity compared with growing at 18-22°C. (Although, this is usually not important in mammalian cells where chaperones compensate for thermolability of Ppy luc2).

3) Comparison of Nanoluc, Antares vs Fluc in live cells, tissues and animals.

What would be the difference when corrected for protein and mRNA half-life - essentially, for actual protein levels in cells during recording/imaging? For example, Fluc luc2 has a protein half-life of 5-8 hours while NanoLuc is much more stable.

4) Do you know what are the actual concentrations of Nanoluc, Fluc, etc in live mammalian cells in relevant experiments (in μM, for example)? So far it seems like no one knows, even Keith Wood himself. :(

On 2017 Mar 06, Misha Koksharov commented:

A really nice FP tool and screening/engineering work.

I have a few questions:

1) "Bioluminescence characterization in vitro. Each protein was diluted to 2 nM in 30mM MOPS 100mM KCl pH 7.5 with 0.1% BSA, and a final concentration of 20 μM substrate was used."

a) Do you mean you have used 20 μM for both LH2 and ATP in case of firefly luciferase (Ppy luc2)?

b) Was Mg²⁺ added to the reaction buffer in case of Fluc?

2) "Hexahistidine-tagged proteins were expressed in E. coli at 30°C for 30 h"

How active was Fluc after growing E.coli at 30°C? I'd expect that this should noticeably decrease its specific activity compared with growing at 18-22°C. (Although, this is usually not important in mammalian cells where chaperones compensate for thermolability of Ppy luc2).

3) Comparison of Nanoluc, Antares vs Fluc in live cells, tissues and animals.

What would be the difference when corrected for protein and mRNA half-life - essentially, for actual protein levels in cells during recording/imaging? For example, Fluc luc2 has a protein half-life of 5-8 hours while NanoLuc is much more stable.

4) Do you know what are the actual concentrations of Nanoluc, Fluc, etc in live mammalian cells in relevant experiments (in μM, for example)? So far it seems like no one knows, even Keith Wood himself. :(