Firefly Luciferase Mutant with Enhanced Activity and Thermostability
نویسندگان
چکیده
منابع مشابه
SpyTag/SpyCatcher Cyclization Enhances the Thermostability of Firefly Luciferase
SpyTag can spontaneously form a covalent isopeptide bond with its protein partner SpyCatcher. Firefly luciferase from Photinus pyralis was cyclized in vivo by fusing SpyCatcher at the N terminus and SpyTag at the C terminus. Circular LUC was more thermostable and alkali-tolerant than the wild type, without compromising the specific activity. Structural analysis indicated that the cyclized LUC i...
متن کاملEndotoxin assay by bioluminescence using mutant firefly luciferase.
The Limulus reaction is an application of the defense mechanism of horseshoe crab for endotoxin detection. Endotoxin is a component of the cell wall in the outer membrane of gram-negative bacteria, and causes fever or shock when it enters the human blood stream. For endotoxin detection, gel formation or turbidity of the coagulation factor chromogen or fluorescence-modified peptide is used. Howe...
متن کاملImproved Red-emitting Firefly Luciferase Mutant for Biotechnical Applications
p 3 Introduction pgs 5-10 Materials and Methods pgs 10-18 Materials General Methods Site-directed Mutagenesis Insertion of Promega’s CBRluc into the pGEX-6P-2 vector Protein Expression and Purification Bioluminescence activity-based light assays Heat inactivation studies Bioluminescence emission spectra Microplate luminometer assays Mammalian cell experiments by Promega collaborators: -Transfec...
متن کاملImproved thermostability of the North American firefly luciferase: saturation mutagenesis at position 354.
We have used random chemical mutagenesis and a simple genetic screen to generate and isolate a thermostable mutant of luciferase from the North American firefly (Photinus pyralis). A single G-to-A transition mutation, resulting in the substitution of a glutamate for a lysine residue at position 354 in the protein sequence, was shown to be responsible for this enhanced thermostability. Replaceme...
متن کاملRegulatory effects of ATP and luciferin on firefly luciferase activity.
ATP and luciferin are not only substrates of firefly luciferase, but can, in addition, modulate its activity. High concentrations of luciferin induce a conformational change of the enzyme that temporarily reduces the catalytic rate. Re-activation takes approx. 20 min and is independent of variation in the concentration of enzyme or ATP, but lengthens with increasing luciferin concentration. Hig...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
ژورنال
عنوان ژورنال: ACS Omega
سال: 2018
ISSN: 2470-1343,2470-1343
DOI: 10.1021/acsomega.7b02068