Luciferase protection against proteolytic degradation: a key for improving signal in nano-system biology.
Luciferase is most widely used bioluminescence protein in biotechnological processes, but the enzyme is susceptible to proteolytic degradation, thereby its intracellular half-life decreased. Osmolytes are known to enhance the stability of proteins and protect them in a native folded and functional state. The effects of osmolytes, including sucrose, glycine and DMSO on the stability of luciferase were investigated. To different extents, all osmolytes protected the luciferase towards proteolytic degradation in a concentration-dependent manner. The results showed that 1.5M sucrose, 1.5M glycine and 15% DMSO are the best. The ability of these osmolytes to protect luciferase against proteolysis decreased from sucrose, glycine, and finally DMSO. Enzymatic kinetic data showed that the luciferase activity is significantly kept in the presence of sucrose and glycine compared to DMSO, particularly at high temperatures. Bioluminescence intensity, circular dichroism (CD), intrinsic and ANS fluorescence experiments showed change in secondary and tertiary luciferase structure. These results suggest that osmolytes exert an important effect on stabilization of luciferase conformation; decreasing the unfolding rate, preventing adaptation and binding of luciferase at the active site of proteases, thereby the proteolytic digestion reduced and its active conformation was kept.
Ataei
Farangis
F
Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
Hosseinkhani
Saman
S
Khajeh
Khosro
K
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D017930:Genes, Reporter; D005998:Glycine / Q000494:pharmacology; D007700:Kinetics; D008156:Luciferases / Q000737:chemistry / Q000378:metabolism*; D036103:Nanotechnology; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D013395:Sucrose / Q000494:pharmacology; D049490:Systems Biology / Q000379:methods*; D013696:Temperature; D014357:Trypsin / Q000494:pharmacology
D000817:Anilino Naphthalenesulfonates; D013395:Sucrose; C027132:1-anilino-8-naphthalenesulfonate; D008156:Luciferases; D014357:Trypsin; D005998:Glycine