Biophysical characterization of highly active recombinant Gaussia luciferase expressed in Escherichia coli.
Recently, the smallest bioluminescent protein (MW: 19.9 kDa), Gaussia luciferase (GLuc), has been isolated from the marine copepod Gaussia princeps and has attracted much attention as a reporter protein. However, preparation of large quantities of homogeneous natively folded recombinant GLuc appears to be difficult due to its ten cysteines. Here, we report the biophysical characterization of recombinant GLuc expressed using a novel Escherichia coli expression system based on a cold induced expression vector (pCold). Using this system, a large fraction of the protein was expressed in the soluble fraction. GLuc, purified exclusively from the supernatant using nickel affinity chromatography, yielded a large amount of pure GLuc with a native disulfide bond pattern (Soluble-GLuc). Soluble-GLuc had a strong bioluminescence activity and it retained 65% of its activity after 30 min incubation at 95 degrees C. Soluble-GLuc remained fully folded until 40 degrees C, as assessed by circular dichroism; and the thermal denaturation curve was S-shaped, indicating a cooperative transition, with a midpoint temperature of 56 degrees C. These results indicate that both the structure and bioluminescence activity of GLuc remain stable at high temperatures, and they strongly suggest GLuc's potential as a reporter protein.
Rathnayaka
Tharangani
T
Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei-shi, Tokyo 184-8588, Japan.
Tawa
Minako
M
Sohya
Shihori
S
Yohda
Masafumi
M
Kuroda
Yutaka
Y
D000818:Animals; D001703:Biophysics; D002942:Circular Dichroism; D033342:Copepoda / Q000201:enzymology* / Q000235:genetics; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008156:Luciferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D004220:Disulfides; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D008156:Luciferases