Abstract
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 [5×105 vs 2.9×106 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 (2.7×107 RLU/mg total protein). These results show promise toward the potential development of an autonomous light generating lux reporter system in mammalian cells
Similar content being viewed by others
References
Almashanu S, Musafia B, Hader R, Suissa M, Kuhn J (1990) Fusion of luxA and luxB and its expression in Escherichia coli, Saccharomyces cerevisiae and Drosophila melanogaster. Biolum Chemilum 5:89–98
Arregui C, Juarez V, zur Hausen H (2003) A synthetic E7 gene of human papillomavirus type 16 that yields enhanced expression of the protein in mammalian cells and is useful for DNA immunization studies. J Virol 77:4928–4937
Baev D, Li X, Edgerton M (2001) Genetically engineered human salivary histatin genes are functional in Candida albicans; development of a new system for studying histatin candidacidal activity. Microbiology 147:3323–3334
Contag PR, Olomu IN, Stevenson DK, Contag CH (1998) Bioluminescent indicators in living mammals. Nat Med 4:245–247
Costa S, Douville E, Bell J, Meighen E (1991) Expression of fused bacterial luciferase in mammalian cells. Biolum Chemilum 1991:31–34
Disbrow GL, Sunitha I, Baker CC, Hanover J, Schlegel R (2003) Codon optimization of the HPV-16 E5 gene enhances protein expression. Virology 311:105–114
Escher A, O’Kane DJ, Lee J, Szalay AA (1989) Bacterial luciferase αβ fusion protein is fully active as a monomer and highly sensitive in vivo to elevated temperature. Proc Natl Acad Sci USA 86:6528–6532
Gelmini S, Pinzani P, Orlando C, Sestini R, Baldwin TO, Pazzagli M (1993) Chloramphenicol acetyl-transferase, firefly and bacterial luciferase as reporter genes in transfection of mammalian cells. Biolum Chemilum 1993:200–206
Guignard I, Combadiere C, Tiffany HL, Murphy PM (1998) Gene organization and promoter function for CC chemokine receptor 5 (CCR5). J Immunol 160:985–992
Gruber MG, Wood KV (2000) Design strategy for synthetic luciferase reporter genes. In: Abstracts for the international symposium for ISBC. Monterey, Calif.
Ichiki T, Usui M, Kato M, Funakoshi Y, Ito K, Egashira K, Takeshita A (1998) Down regulation of angiotensin II type 1 receptor gene transcription by nitric oxide. Hypertension 31:342–348
Kirchner G, Roberts JL, Gustafson GD, Ingolia TD (1989) Active bacterial luciferase from a fused gene expression of a Vibrio harveyi luxAB translational fusion in bacteria, yeast and plant cells. Gene 81:349–354
Kurland CG (1991) Codon bias and gene expression. FEBS Lett 285:165–169
Narum DL, Kumar S, Rogers WO, Fuhrmann SR, Liang H, Oakley M, Taye A, Sim BK, Hoffman SL (2001) Codon optimization of gene fragments encoding Plasmodium falciparum merzoite proteins enhances DNA vaccine protein expression and immunogenicity in mice. Infect Immun 69:7250–7253
Naylor LH (1999) Reporter gene technology: the future looks bright. Biochem Pharm 58:749–757
Olsson O, Escher A, Sandberg G, Schell J, Koncz C, Szalay AA (1989) Engineering of monomeric bacterial luciferases by fusion of luxA and luxB genes in Vibrio harveyi. Gene 81:335–347
Pazzagli LJ, Devine H, Peterson DO, Baldwin TO (1992) Use of bacterial and firefly luciferase as reporter genes in DEAE-dextran-mediated transfection in mammalian cells. Biochemistry 204:315–323
Prodromou C, Pearl LH (1992) Recursive PCR: a novel technique for total gene synthesis. Prot Eng 5:827–829
Schwechheimer C, Smith C, Bevan MW (1998) The activities of acidic and glutamine-rich transcriptional activation domains in plant cells: Design of modular transcription factors for high-level expression. Plant Mol Biol 36:195–204
Watson MA, Darrow C, Zimonjic DB, Popescu NC, Fleming TP (1998) Structure and transcriptional regulation of the human mammaglobin gene, a breast cancer associated member of the uteroglobin gene family localized to chromosome 11q13. Oncogene 16:817–824
Zhang J, Kuvelkar R, Cheewatrakoolpong B, Williams S, Egan RW, Billah MM (1987) Evidence for multiple promoters of the human IL-5 receptor α subunit gene. A novel 6-base pair element determines cell-specific promoter function. J Immunol 159:5412–5421
Zhang T, Kruys V, Huez G, Gueydan C (2002) AU-rich element-mediated translational control: complexity and multiple activities of trans-activating factors. Biochem Soc Trans 30:952–958
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Patterson, S.S., Dionisi, H.M., Gupta, R.K. et al. Codon optimization of bacterial luciferase (lux) for expression in mammalian cells. J IND MICROBIOL BIOTECHNOL 32, 115–123 (2005). https://doi.org/10.1007/s10295-005-0211-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10295-005-0211-8