Expression, Purification and Biophysical Characterization of ATP Independent Luciferase From Gaussia princeps
The determination of the three dimensional structure of the GLuc-protein is needed to further explore the underlying reaction mechanism. In this work, GLuc has been cloned, heterologically expressed in E. coli cells, and a protocol was established to purify the full-length construct to homogeneity, as judged by SDS-PAGE. Furthermore, characterization of the soluble protein was performed employing the following biophysical methods: dynamic light scattering, high pressure liquid chromatography, nuclear magnetic resonance and biolayer interferometry. The expression and purification from the optimised protocol produce a soluble recombinant luciferase enzyme with high bioluminescence activity and stability. The tested initial crystallization conditions produced small needle clusters and some spherulites. This thesis confirms that luciferase consists of two structural domain repeats as deduced from the cDNA sequence. Both domains, when expressed separately, have bioluminescence activity with CTZ. The catalytic properties of both domains were characterized in more detail. For this purpose, the full-length GLuc with a thrombin cleavage site between both domains, and the two individual catalytic domains were cloned and expressed in E. coli. The proteins were purified by gel filtration and the results were analysed by SDS-PAGE and HPLC. Both domains were found to require identical buffer conditions for maximal light yield. The catalytic properties including optimal buffer condition (pH, temperature, salt and detergent concentration) are identical to the intact full-length Gaussia luciferase. Besides the construction of the individual full-length GLuc construct for expression in E. coli, three constructs were generated for expression in Sf21 insect cells using the baculovirus expression vector system (BEVS) for protein crystallization in living cells. The results show the formation of GLuc microcrystals in vivo, which were visualized with a light microscope.