A set of multicolored Photinus pyralis luciferase mutants for in vivo bioluminescence applications

A set of multicolored Photinus pyralis luciferase mutants for in vivo bioluminescence applications.

Error-prone PCR was used to isolate Photinus pyralis luciferase mutants producing bright light in the red-orange region of the spectrum. All mutations were clustered in the beta5-alpha10-beta6 region of N-terminal subdomain B and appear to affect bioluminescence color by modulating the position of the Ser314-Leu319 mobile loop with respect to the putative active site. Two red variants (Q283R an...

Development of a pH-Tolerant Thermostable Photinus pyralis Luciferase for Brighter In Vivo Imaging

Mutagenesis of solvent-exposed amino acids in Photinus pyralis luciferase improves thermostability and pH-tolerance.

Firefly luciferase catalyses a two-step reaction, using ATP-Mg2+, firefly luciferin and molecular oxygen as substrates, leading to the efficient emission of yellow-green light. We report the identification of novel luciferase mutants which combine improved pH-tolerance and thermostability and that retain the specific activity of the wild-type enzyme. These were identified by the mutagenesis of ...

Equilibrium intermediates in the reversible unfolding of firefly (Photinus pyralis) luciferase.

Firefly luciferase has been used as a model protein to study cotranslational and chaperone-assisted protein folding. We found conditions for reversible unfolding of luciferase in the absence of cellular factors, and we characterized denaturant-induced equilibrium unfolding transitions and refolding kinetics of the enzyme. Luciferase unfolding induced by guanidinium chloride at 10 degrees C can ...

Measurement of Michaelis Constants for ATP and Mg2+ in Bioluminescence Reaction of Luciferase by a Home-Made Luminometer

Effects of ATP and Mg2+ concentrations on bioluminescence reaction of luciferase (Photinus pyralis) were investigated by home-made luminometer. The Michaelis constants of the enzyme for ATP and Mg2+ obtained from the Lineweaver-Burk graph, were 61.9 mM ±3.3 mM and 251.6mM ± 39.0mM...