Violet and greenish photoprotein obelin mutants for reporter applications in dual-color assay

Two kinds of Ca2+-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 Ca2+ 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.