Rapidly maturing red fluorescent protein variants with strongly enhanced brightness in bacteria.
A rapidly maturing variant of the red fluorescent protein DsRed was optimized for bacterial expression by random mutagenesis. The brightest variant contains six mutations, two of which (S4T and a silent mutation in codon 2) explain most of the fluorescence enhancement. The novel variants are expressed at 9-60-fold higher levels in Escherichia coli compared to DsRed.T3, but are not superior fluorophores on a per molecule basis. In contrast to previously available DsRed variants, DsRed.T3_S4T is sufficiently bright to monitor Salmonella gene expression in infected animals using flow cytometry. However, no fluorescence enhancement was observed in Leishmania or HeLa cells, indicating that these novel variants are specifically useful for bacteria.
Sörensen
Meike
M
Department of Molecular Biology, Max-Planck-Institute for Infection Biology, Schumannstr. 21/22, D-10117 Berlin, Germany.
Lippuner
Christoph
C
Kaiser
Toralf
T
Misslitz
Ana
A
Aebischer
Toni
T
Bumann
Dirk
D
D000818:Animals; D001483:Base Sequence; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005260:Female; D005453:Fluorescence; D014644:Genetic Variation; D006367:HeLa Cells; D006801:Humans; D007894:Leishmania mexicana / Q000235:genetics; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D018613:Microscopy, Confocal; D016296:Mutagenesis; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012475:Salmonella / Q000235:genetics; D000097573:Red Fluorescent Protein
D004274:DNA, Recombinant; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D000097573:Red Fluorescent Protein