Author:
Mitani Yasuo,Yasuno Rie,Kihira Kiyohito,Chung KwiMi,Mitsuda Nobutaka,Kanie Shusei,Tomioka Azusa,Kaji Hiroyuki,Ohmiya Yoshihiro
Abstract
Cypridina noctiluca luciferase (CLuc) is a secreted luminescent protein that reacts with its substrate (Cypridina luciferin) to emit light. CLuc is known to be a thermostable protein and has been used for various research applications, including in vivo imaging and high-throughput reporter assays. Previously, we produced a large amount of recombinant CLuc for crystallographic analysis. However, this recombinant protein did not crystallize, probably due to heterogeneous N-glycan modifications. In this study, we produced recombinant CLuc without glycan modifications by introducing mutations at the N-glycan modification residues using mammalian Expi293F cells, silkworms, and tobacco Bright Yellow-2 cells. Interestingly, recombinant CLuc production depended heavily on the expression hosts. Among these selected hosts, we found that Expi293F cells efficiently produced the recombinant mutant CLuc without significant effects on its luciferase activity. We confirmed the lack of N-glycan modifications for this mutant protein by mass spectrometry analysis but found slight O-glycan modifications that we estimated were about 2% of the ion chromatogram peak area for the detected peptide fragments. Moreover, by using CLuc deletion mutants during the investigation of O-glycan modifications, we identified amino acid residues important to the luciferase activity of CLuc. Our results provide invaluable information related to CLuc function and pave the way for its crystallographic analysis.
Funder
Japan Society for the Promotion of Science
Subject
Biomedical Engineering,Histology,Bioengineering,Biotechnology
Reference35 articles.
1. BryanB. J.
Szent-GyorgyiC.
Pittsburgh, PAProlume, LTDLuciferases, Fluorescent Proteins, Nucleic Acids Encoding the Luciferases and Fluorescent Proteins and the Use Thereof in Diagnostics, High Throughput Screening and novelty Items2001
2. Post-expression Strategies for Structural Investigations of Membrane Proteins;Columbus;Curr. Opin. Struct. Biol.,2015
3. Three-dimensional Structure of Human Follicle-Stimulating Hormone;Fox;Mol. Endocrinol.,2001
4. Engineered Luciferase Reporter from a Deep Sea Shrimp Utilizing a Novel Imidazopyrazinone Substrate;Hall;ACS Chem. Biol.,2012
5. Intracellular Functions of N-Linked Glycans;Helenius;Science,2001
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