New mutated photoproteins derived from jellyfish photoproteins have increased thermostability or increased luminescence time and are useful as bioluminescent markers, e.g., to detect pathogens

摘要

Mutated photoproteins derived from photoproteins isolated from jellyfish, where the mutations give increased thermostability and/or increased luminescence time are new. Mutated photoproteins derived from photoproteins isolated from jellyfish, where the mutations give increased thermostability in the cell and/or increased luminescence time. Increased thermostability is achieved by at least one of the following mutations with the motif being conserved: (a) suppression of lysine by substitution with arginine in the motif Arg-His-Lys-X1-Met-Phe-X2 where X1 = is His or Phe X2 = Asn or Asp (b) suppression of glutamine by substitution with arginine in the motif Asp-Glu-Met-Thr-Arg-Gln-His-Leu-Gly-Phe-Trp-Tyr; (c) suppression of leucine by substitution with isoleucine in the motif Asp-Glu-Met-Thr-Arg-Gln-Gly-His-Leu-Gly-Phe-Trp-Tyr. Increased luminescence time is achieved by at least one of the following mutations with the motif being conserve: (a) suppression of glutamine by glutamate or glycine in the motif Asp-X1-Asn-X2X3-Gly-X4-Ile-X5-Leu-X6-Glu where X1 = Val or Ile X2 = His,Gly or Ser X3 = Asn or Asp X4 = Lys or Glu X5 = Ser, Thr or His X6 = Asp or Asn (b) suppression of aspartate by substitution with glycine in the motif Asp-Lys-Asp-X1X2-Gly-X3X4X5-Leu-Asp-Glu where X1 = Gln, Gly or Arg X2 = Asn or Ser X3 = Ala, Ser or Thr X4 = Ile or Val X5 = Thr or Ser (c) suppression of glutamine by substitution with glycine in the motif Asp-Lys-Asp-X1X2-Gly-X3X4X5-Leu-Asp-Glu; (d) suppression of aspartate by substitution with glycine in the motif Asp-X1X2-Gly-X3X4-Leu-Asp-Val-Asp-Glu where X1 = Ile or Leu X2 = Glu, Asn or Gly X3 = Ser or Asp X4 = Gln, Lys or Asp (e) suppression of phenylalanine by substitution with serine in the motif Glu-X1Thr-Phe-X2X3 where X1 = Glu, Lys or Ala X2 = Arg, Lys or Ala X3 = Val or His (f) suppression of valine at position 54 of the 199 amino acid sequence fully defined in the specification by substitution with alanine. Independent claims are also included for the following: (i) nucleotide sequences encoding the claimed photoproteins; (ii) a vector, for example a plasmid containing the nucleotide sequences; (iii) host cells particularly E. coli, HEK 293 or CHO cells transformed with the above vector; (iv) preparing mutated the photoproteins comprising transforming a host cell with the above vector and purifying the produced photoproteins; (v) detecting in vitro proteins, antigens or nucleic acids in a biological sample, comprising if necessary amplifying nucleic acids in the sample, immobilizing the antigens, proteins or nucleic acids on a support, adding an appropriate probe with the photoprotein attached, rinsing off unattached probe and measuring bioluminescence intensity; (vi) detecting enzymatic activity in vitro in a biological sample, comprising immobilizing on a support a substrate specific for the enzyme to be detected linked to a photoprotein, and measuring bioluminescence intensity after rinsing; and (vii) detecting variations in intracellular calcium induced by various agents, comprising cultivating the claimed transformed host cells with a sample and measuring variation in bioluminescence