Codon optimization of bacterial luciferase (lux) for expression in mammalian cells.
Expression of the bacterial luciferase (lux) system in mammalian cells would culminate in a new generation of bioreporters for in vivo monitoring and diagnostics technology. Past efforts to express bacterial luciferase in mammalian cells have resulted in only modest gains due in part to low overall expression of the bacterial genes. To optimize expression, we have designed and synthesized codon-optimized versions of the luxA and luxB genes from Photorhabdus luminsecens. To evaluate these genes in vivo, stable HEK293 cell lines were created harboring wild type luxA and luxB (WTA/WTB), codon-optimized luxA and wild type luxB (COA/WTB), and codon-optimized versions of both luxA and luxB genes (COA/COB). Although mRNA levels within these clones remained approximately equal, LuxA protein levels increased significantly after codon optimization. On average, bioluminescence levels were increased by more than six-fold [5x10(5) vs 2.9x10(6) relative light units (RLU)/mg total protein] with the codon-optimized luxA and wild type luxB. Bioluminescence was further enhanced upon expression of both optimized genes (2.7x10(7) RLU/mg total protein). These results show promise toward the potential development of an autonomous light generating lux reporter system in mammalian cells.
Patterson
Stacey S
SS
Department of Microbiology, The University of Tennessee, Knoxville, TN 37996, USA.
Dionisi
Hebe M
HM
Gupta
Rakesh K
RK
Sayler
Gary S
GS
D000818:Animals; D001483:Base Sequence; D001709:Biotechnology / Q000379:methods*; D002460:Cell Line; D003062:Codon / Q000235:genetics*; D015870:Gene Expression; D017930:Genes, Reporter / Q000235:genetics*; D006801:Humans; D007668:Kidney / Q000166:cytology; D008156:Luciferases / Q000032:analysis / Q000235:genetics*; D008322:Mammals; D008967:Molecular Biology / Q000379:methods*; D008969:Molecular Sequence Data; D009876:Operon; D012333:RNA, Messenger / Q000032:analysis
D003062:Codon; D012333:RNA, Messenger; D008156:Luciferases