Bacterial luciferase alpha beta fusion protein is fully active as a monomer and highly sensitive in vivo to elevated temperature.
A 2.2-kilobase-pair (kbp) DNA fragment from Vibrio harveyi contains the luxA and luxB genes separated by a 26-base-pair (bp) intergenic region. The two genes were converted to a single open reading frame by site-specific mutagenesis. A full-length fusion protein is obtained when the new gene is placed under transcriptional control of a T7 promoter in Escherichia coli. Bioluminescence of colonies containing the gene fusion is 0.002% of the wild-type luciferase [alkanal monooxygenase (FMN-linked); alkanal, reduced-FMN:oxygen oxidoreductase (1-hydroxylating, luminescing), EC 1.14.14.3] at 37 degrees C. Growth at 23 degrees C results in a greater than 50,000-fold increase in light emission in cells containing fusion protein, whereas only a 3-fold increase in observed with cells containing the luxAB dicistron. Purified fusion protein isolated from E. coli grown at 19 degrees C exists in both monomeric and dimeric forms with specific bioluminescence activities comparable to the heterodimeric wild-type enzyme at 23 degrees C and 37 degrees C. These findings show that the alpha beta fusion polypeptide is functional as a monomer and suggest that its folding is drastically affected at elevated temperature. We hypothesize that the two-subunit bacterial luciferase may have evolved from a monomer as a result of a temperature increase in the environment.
Escher
A
A
Department of Cell Biology, University of Alberta, Edmonton, Canada.
O'Kane
D J
DJ
Lee
J
J
Szalay
A A
AA
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D005798:Genes, Bacterial; D007438:Introns; D008156:Luciferases / Q000235:genetics* / Q000378:metabolism; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013816:Thermodynamics; D014733:Vibrio / Q000201:enzymology / Q000235:genetics*
D011993:Recombinant Fusion Proteins; D008156:Luciferases