Genetically encoded bioluminescent indicators for stress hormones.
This study demonstrates bioluminescent indicators for determining stress hormones in mammalian cells. A genetically encoded bioluminescent probe for stress sensing was first synthesized with a LXXLL motif-linked ligand binding domain of the glucocorticoid receptor (GR LBD), which was then sandwiched between the fragments of Gaussia luciferase (GLuc). This prototype of the bioluminescent indicators was carefully modified with a circular permutation (CP) and/or a corepressor motif. The first notable appearance by cofusion of a corepressor motif to the probe was the biphasic dose-response curves of the indicator to cortisol. A CP largely improved the detection limit of the indicator to cortisol up to 100 times. Fabrication of both CP and the attachment of a corepressor motif in the indicator synergistically contributes to (i) the lower detection limit and wider dynamic range and (ii) the enhanced absolute luminescence and ligand selectivity. This study is the first example that contribution of corepressor motifs to single-chain probes was investigated. This study also provides new insight into improving the sensorial properties of single-chain probes with CP.
Kim
Sung Bae
SB
National Institute of Advanced Industrial Science and Technology, 16-1 Onogawa, Tsukuba 305-8569, Japan.
Sato
Moritoshi
M
Tao
Hiroaki
H
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D006854:Hydrocortisone / Q000032:analysis* / Q000652:urine; D008156:Luciferases / Q000235:genetics* / Q000378:metabolism; D049408:Luminescent Agents / Q000737:chemistry*; D008163:Luminescent Measurements / Q000379:methods*; D017434:Protein Structure, Tertiary; D011965:Receptors, Glucocorticoid / Q000235:genetics* / Q000378:metabolism
D049408:Luminescent Agents; D011965:Receptors, Glucocorticoid; D008156:Luciferases; D006854:Hydrocortisone