Solubilization and folding of a fully active recombinant Gaussia luciferase with native disulfide bonds by using a SEP-Tag.
Biochim Biophys Acta
; 1814(12): 1775-8, 2011 Dec.
Article
em En
| MEDLINE
| ID: mdl-21945374
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Copépodes
/
Dissulfetos
/
Luciferases
Limite:
Animals
Idioma:
En
Ano de publicação:
2011
Tipo de documento:
Article