Team:Hamburg/Proof Of Concept

Proof of concept

To generally prove that the fusion proteins successfully metabolize their specific substrates, gas chromatography, coupled with mass spectrometry (GC-MS) was used. No traces of the desired products, such as α-humulene, amorphadiene nor linear α-olefins, could be detected. The expression of the fusion proteins could be partially proven by western blots with anti-His antibodies. The planned confirmation with matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF) could not be done due to time limitations.

Ideally, because of lack of structures, crystallography experiments for enzyme structure elucidation were planned. To confirm activity of CYP1A1, which metabolizes 7-ethoxycoumarin to 7-hydroxycoumarin, plate reader measurements were performed. Fluorescence at 465 nm was obtained, indicating metabolization. Similar levels of fluorescence were detected in the negative controls, negating the proof of activity. To prove the concept of the softwaretool, LEA was executed with an example sequence to check the similarity of the predicted linker sequences to the actual linker sequences. The high similarity confirmed the concept of LEA. An interesting addition would be looking at the resulting 3D-structures with the newly released AlphaFold software. This approach would be ideal to confirm that the predicted fusion protein sequences produce structurally sensible results.