Violet and greenish photoprotein obelin mutants for reporter applications in dual-color assay.
Two kinds of Ca(2+)-regulated photoprotein obelin with altered color of bioluminescence were obtained by active-center amino acid substitution. The mutant W92F-H22E emits violet light (lambda(max) = 390 nm) and the mutant Y139F emits greenish light (lambda(max) = 498 nm), with small spectral overlap, both display high activity and stability and thus may be used as reporters. For demonstration, the mutants were applied in dual-color simultaneous immunoassay of two gonadotropic hormones-follicle-stimulating hormone and luteinizing hormone. Bioluminescence of the reporters was simultaneously triggered by single injection of Ca(2+) solution, divided using band-pass optical filters and measured with a two-channel photometer. The sensitivity of simultaneous bioluminescence assay was close to that of a separate radioimmunoassay.
Frank
Ludmila A
LA
Institute of Biophysics, Russian Academy of Sciences, Siberian Branch, Krasnoyarsk, 660036, Russia. lfrank@yandex.ru
Borisova
Vasilisa V
VV
Markova
Svetlana V
SV
Malikova
Natalia P
NP
Stepanyuk
Galina A
GA
Vysotski
Eugene S
ES
D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000737:chemistry; D003116:Color; D005640:Follicle Stimulating Hormone / Q000032:analysis; D007118:Immunoassay / Q000379:methods*; D008163:Luminescent Measurements / Q000379:methods; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics*; D007986:Luteinizing Hormone / Q000032:analysis; D017354:Point Mutation; D013997:Time Factors
D008164:Luminescent Proteins; C010497:obelin; D007986:Luteinizing Hormone; D005640:Follicle Stimulating Hormone; D002118:Calcium