Fusion ofluxA andluxB and its expression inE. coli, S. cerevisiae andD. melanogaster
✍ Scribed by Almashanu, S. ;Musafia, B. ;Hadar, R. ;Suissa, M. ;Kuhn, J.
- Book ID
- 102762044
- Publisher
- John Wiley and Sons
- Year
- 1990
- Weight
- 620 KB
- Volume
- 5
- Category
- Article
- ISSN
- 0884-3996
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✦ Synopsis
Luciferase from Vibrio harveyi is encoded by t w o adjacent genes, luxA and IuxB. The t w o genes were fused by replacing a segment extending from near the end o f luxA into the N-terminal end of lux6 by a synthetic oligonucleotide. The construction removed the TAA stop codon at the end o f IuxA, the intervening region of 26 base pairs, and the initial methionine o f lux6. A Smal site was included a t the junction between the t w o genes and an Aatll site was created near the end of luxA without altering its amino acid sequence. In Escherichia coli the fused IuxAB gene could be expressed t o produce functional luciferase that gave about 20% o f the activity in cells without the fusion.
An out-of-frame ATG exists close t o and preceding the ATG of the luxA gene. This was removed and the entire fused gene bracketed by several restriction enzyme sites.
The fused luxA6 gene was successfully expressed in Saccharomyces cerevisiae and Drosophila melanogaster by transferring it t o appropriate plasmid vectors.