title: Efficient production of glycosylated Cypridina luciferase using plant cells.
abstract: Cypridina noctiluca luciferase has been utilized for biochemical and molecular biological applications, including bioluminescent enzyme immunoassays, far-red luminescence imaging, and high-throughput reporter assays. Some of these applications require a large amount of purified luciferase. However, conventional protein expression systems are not capable of producing sufficient quantities of protein with a high quality and purity without laborious and costly purification processes. To improve the productivity and expand the breadth of possibilities for Cypridina luciferase applications, we employed a variety of secretion expression systems, including yeast, mammalian cells, and silk worms. In this study, we established a simple production procedure using plant cell cultures. The plant cell culture BY-2 efficiently secreted luciferase, which was easily purified using a simple one-step ion-exchange chromatography method. The production yield was 20-30 mg of luciferase per liter of culture medium, and its Km for the luciferin (0.45 μM) was similar to that of the native protein. Additionally, we characterized its glycosylation pattern and confirmed that the two potential N-glycosylation sites were modified with plant-type oligosaccharide chains. Interestingly, the oligosaccharide chains could be trimmed without any detectable decrease in recombinant protein activity. Therefore, the results of our study indicate that this method offers a more cost-effective production method for Cypridina luciferase than conventional methods.
authors.affiliation: Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba 305-8566, Japan. Electronic address: mitani-y@aist.go.jp.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Mitani
authors.affiliation: Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba 305-8566, Japan.
authors.forename: Yoshimi
authors.initials: Y
authors.lastname: Oshima
authors.affiliation: Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba 305-8566, Japan.
authors.forename: Nobutaka
authors.initials: N
authors.lastname: Mitsuda
authors.affiliation: Biotechnology Research Institute for Drug Discovery, AIST, Tsukuba 305-8568, Japan.
authors.forename: Azusa
authors.initials: A
authors.lastname: Tomioka
authors.affiliation: Biotechnology Research Institute for Drug Discovery, AIST, Tsukuba 305-8568, Japan.
authors.forename: Masako
authors.initials: M
authors.lastname: Sukegawa
authors.affiliation: Biotechnology Research Institute for Drug Discovery, AIST, Tsukuba 305-8568, Japan.
authors.forename: Mika
authors.initials: M
authors.lastname: Fujita
authors.affiliation: Biotechnology Research Institute for Drug Discovery, AIST, Tsukuba 305-8568, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kaji
authors.affiliation: DAILAB, Biomedical Research Institute, AIST, Tsukuba 305-8566, Japan.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Ohmiya
mesh_terms: D000818:Animals; D017360:Arabidopsis / Q000166:cytology* / Q000378:metabolism*; D060829:Arthropod Proteins / Q000096:biosynthesis / Q000235:genetics; D003445:Crustacea / Q000201:enzymology / Q000235:genetics*; D006031:Glycosylation; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D059828:Plant Cells / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D060829:Arthropod Proteins; D011994:Recombinant Proteins; D008156:Luciferases
keywords: Cypridina luciferase; Glycosylation; Plant cell