Design and characterization of novel trypsin-resistant firefly luciferases by site-directed mutagenesis

Abstract

Firefly luciferase (EC.1.13.12.7) from Photinus pyralis is a single polypeptide chain (62 kDa), responsible for emission of yellow-green (557 nm) light, known to be most efficient bioluminescence system that make it an excellent tool for reporter in nano-system biology. However, it is very sensitive to proteolytic degradation, which reduces its intracellular half-life, leads to loss in sensitivity and precision in analytic applications. In order to generate more stable luciferases against protease digestion, we substituted two tryptic sites: R(213), R(337) and also next residue to it (Q(338)) with another amino acids. Overall, all mutations brought about structural changes that indicated more compact structure upon mutation, which revealed by enhancement of tryptophan fluorescence, decreases flexibility and less surface hydrophobic pockets. In general, structural changes associated with a clear improvement in thermostability and resistance against trypsin hydrolysis. In particular, R337Q mutant shows higher light stability in mammalian cell culture, which makes it as a suitable reporter for imaging.

The role of proline substitutions within flexible regions on thermostability of luciferase

“…The most widely used luciferase reporter gene is that of Photinus pyralis (P. pyralis, North American Firefly). Wild-type P. pyralis luciferase is thermolabile, with an in vitro half-life for activity of the order of 2-3 min at 37°C, which limits the wide industrial applications of this enzyme [2]. The design of proteins with enhanced thermostability is one of the major goals of protein engineering.…”

Effects of Sucrose and Trehalose on Stability, Kinetic Properties, and Thermal Aggregation of Firefly Luciferase

“…Previously, thermostable mutants have been designed to improve the utilization and thermostability of firefly luciferase [27][28][29][30][31]. Moreover, osmolytes as general stabilizing additives have been used to increase the stability of firefly luciferase at higher temperatures [32][33][34].…”

“…Moreover, osmolytes as general stabilizing additives have been used to increase the stability of firefly luciferase at higher temperatures [32][33][34]. Firefly luciferase is a thermosensitive protein that usually loses its activity at 37°C in 3 min, but it keeps its activity in vivo for 4-5 h [30,31].…”

Molecular enigma of multicolor bioluminescence of firefly luciferase

“…Among those, matrix protein and protein-protein interactions, the presence of proteases may be mentioned, as their effect on luciferase structural stability and flexibility has been shown [114][115][116]. Decrease of protease-prone regions of firefly luciferase by site-directed mutagenesis brought about with more structural stability, higher and more stable bioluminescence signal in cell culture [117].…”