Fusion of LuxA and LuxB and its expression in E. coli, S. cerevisiae and D. melanogaster.
Luciferase from Vibrio harveyi is encoded by two adjacent genes, luxA and luxB. The two genes were fused by replacing a segment extending from near the end of luxA into the N-terminal end of luxB by a synthetic oligonucleotide. The construction removed the TAA stop codon at the end of luxA, the intervening region of 26 base pairs, and the initial methionine of luxB. A Smal site was included at the junction between the two genes and an AatII site was created near the end of luxA without altering its amino acid sequence. In Escherichia coli the fused luxAB gene could be expressed to produce functional luciferase that gave about 20% of the activity in cells without the fusion. An out-of-frame ATG exists close to and preceding the ATG of the luxA gene. This was removed and the entire fused gene bracketed by several restriction enzyme sites. The fused luxAB gene was successfully expressed in Saccharomyces cerevisiae and Drosophila melanogaster by transferring it to appropriate plasmid vectors.
Almashanu
S
S
Faculty of Biology, Technion-Israel Institute of Technology, Haifa.
Musafia
B
B
Hadar
R
R
Suissa
M
M
Kuhn
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004331:Drosophila melanogaster / Q000201:enzymology / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D008156:Luciferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D014733:Vibrio / Q000201:enzymology / Q000235:genetics*
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D008156:Luciferases