title: Solubilization and folding of a fully active recombinant Gaussia luciferase with native disulfide bonds by using a SEP-Tag.
abstract: Gaussia luciferase (GLuc) is the smallest known bioluminescent protein and is attracting much attention as a potential reporter protein. However, its 10 disulfide bond forming cysteines have hampered the efficient production of recombinant GLuc and thus limited its use in bio-imaging application. Here, we demonstrate that the addition of a short solubility enhancement peptide tag (SEP-Tag) to the C-terminus of GLuc (GLuc-C9D) significantly increased the fraction of soluble protein at a standard expression temperature. The expression time was much shorter, and the final yield of GLuc-C9D was significantly higher than with our previous pCold expression system. Reversed phase HPLC indicated that the GLuc-C9D variant folded with a single disulfide bond pattern after proper oxidization. Further, the thermal denaturation of GLuc-C9D was completely reversible, and its secondary structure content remained unchanged until 40°C as assessed by CD spectroscopy. The (1)H-NMR spectrum of GLuc indicated sharp well dispersed peaks typical for natively folded proteins. GLuc-C9D bioluminescence activity was strong and fully retained even after incubation at high temperatures. These results suggest that solubilization using SEP-Tags can be useful for producing large quantities of proteins containing multiple disulfide bonds.
authors.affiliation: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei-shi, Tokyo, Japan.
authors.forename: Tharangani
authors.initials: T
authors.lastname: Rathnayaka
authors.forename: Minako
authors.initials: M
authors.lastname: Tawa
authors.forename: Takashi
authors.initials: T
authors.lastname: Nakamura
authors.forename: Shihori
authors.initials: S
authors.lastname: Sohya
authors.forename: Kunihiro
authors.initials: K
authors.lastname: Kuwajima
authors.forename: Masafumi
authors.initials: M
authors.lastname: Yohda
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Kuroda
mesh_terms: D000818:Animals; D003001:Cloning, Molecular; D033342:Copepoda / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry*; D004789:Enzyme Activation; D017930:Genes, Reporter; D008156:Luciferases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D009842:Oligopeptides / Q000235:genetics; D015202:Protein Engineering; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012995:Solubility
chemical_list: D004220:Disulfides; D008164:Luminescent Proteins; D009842:Oligopeptides; D011994:Recombinant Proteins; C037841:tyrosyl-alanyl-glycine; D008156:Luciferases