Development of a thermostable firefly luciferase

Tisi, Laurence; White, Peter J; Squirrell, D.J.; Murphy, Melanie; Lowe, Christopher R.; Murray, James A. H.

doi:10.1016/s0003-2670(01)01496-9

Cited by 57 publications

(42 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Although thermolability is advantageous for time-sensitive studies, such as induction or repression of gene expression, drug response etc., a more thermostable enzyme results in accumulation of more enzyme over time, thus producing much higher light output. Tisi et al (13) thus constructed a thermostable fl (tfl bearing the mutations E354K, I232A, T214A, and F295L), which has an in vitro half-life of 15 min. However, the COOH-terminal end of the tfl contains a peroxisome targeting sequence [serine-lysine-leucine (SKL)] that directs the enzyme to the peroxisome (14).…”

Section: Introductionmentioning

confidence: 99%

Construction and Validation of Improved Triple Fusion Reporter Gene Vectors for Molecular Imaging of Living Subjects

2007

View full text Add to dashboard Cite

Multimodality imaging using several reporter genes and imaging technologies has become an increasingly important tool in determining the location(s), magnitude, and time variation of reporter gene expression in small animals. We have reported construction and validation of several triple fusion genes composed of a bioluminescent, a fluorescent, and a positron emission tomography (PET) reporter gene in cell culture and in living subjects. However, the bioluminescent and fluorescent components of fusion reporter proteins encoded by these vectors possess lesser activities when compared with the bioluminescent and fluorescent components of the nonfusions. In this study, we first created a mutant (mtfl) of a thermostable firefly luciferase (tfl) bearing the peroxisome localization signal to have greater cytoplasmic localization and improved access for its substrate, D-luciferin. Comparison between the three luciferases [mtfl, tfl, and firefly luciferase ( fl)] both in cell culture and in living mice revealed that mtfl possessed 6-to 10-fold (in vitro) and 2-fold (in vivo) higher activity than fl. The improved version of the triple fusion vector carrying mtfl as the bioluminescent reporter component showed significantly (P < 0.05) higher bioluminescence than the previous triple fusion vectors. Of the three different red fluorescent reporter genes ( jred, hcred, and mrfp1, isolated from jellyfish chromophore, coral Heteractis crispa, and coral Discosoma, respectively) evaluated, mrfp1 was able to preserve highest expression as a component of the triple fusion reporter gene for in vivo fluorescence imaging. A truncated version of wild-type herpes simplex virus 1 (HSV1) thymidine kinase gene (wttk) retained a higher expression level than the truncated mutant HSV1-sr39 TK (ttk) as the third reporter component of this improved triple fusion vector. Multimodality imaging of tumor-bearing mice using bioluminescence and microPET showed higher luciferase activity [(2.7 F 0.1 versus 1.9 F 0.1) Â (10 6 p/s/cm 2 /sr)] but similar level of fluorine-18-labeled 2 ¶-fluoro-2 ¶-deoxyarabinofuranosyl-5-ethyluracil (18F-FEAU) uptake (1.37 F 0.15 versus 1.37 F 0.2) percentage injected dose per gram] by mtfl-mrfp1-wttk-expressing tumors compared with the fl-mrfp1-wttkexpressing tumors. Both tumors showed 4-to 5-fold higher accumulation (P < 0.05) of 18F-FEAU than fluorine-18-labeled 9-(4-fluoro-3-hydroxymethylbutyl)guanine. This improved triple fusion reporter vector will enable high sensitivity detection of lower numbers of cells from living animals using the combined bioluminescence, fluorescence, and microPET imaging techniques. [Cancer Res 2007;67(7):3085-93]

show abstract

Section: Introductionmentioning

confidence: 99%

Construction and Validation of Improved Triple Fusion Reporter Gene Vectors for Molecular Imaging of Living Subjects

2007

View full text Add to dashboard Cite

show abstract

“…At the same time even in the solutions with high ionic strength and in the presence of glycerol (10%) and bovine serum albumin (10 mg/ml) P. pyralis luciferase loses >90% of activity in vitro within 6-20 min at 37-42°C (Nguyen et al, 1989;Thulasiraman & Matts, 1996;Tisi et al, 2002;White et al, 1996). Similar results were obtained for L. mingrelica luciferase (Lundovskikh et al, 1998;Ugarova et al, 2000).…”

Section: Stability Of Firefly Luciferases In Solutionmentioning

confidence: 59%

“…Combining these point mutations with the E354K mutation into the P.pyralis gene resulted in mutant luciferase (rLucx4ts) that had an increase in thermostability of about 7°C relative to the wild-type enzyme. Hence, in this case the multiple point mutations led to a cumulative increase in thermostability (Tisi et al, 2002).…”

Section: Thermostabilization Of Firefly Luciferases By Random and Sitmentioning

confidence: 90%

Thermostabilization of Firefly Luciferases Using Genetic Engineering

Ugarova¹,

Koksharov²

2012

Genetic Engineering - Basics, New Applications and Responsibilities

View full text Add to dashboard Cite

“…36 In Ppy, thermostability was increased by the mutation of negatively charged E354 (corresponding to N351 of PhRED) to positively charged K or R 30 and by the combinations of E354K with A215L (217 of Lcr and Lla) or with T214A and I232A. 31 These results indicate the fundamental importance of position 354 in firefly luciferase stability, which is verified by the mutant E356R of Hpa (354 of Ppy). 35 In addition, mutations of solvent-exposed nonconserved hydrophobic residues to hydrophilic residues in Ppy (e.g., F465R: i.e., S463 of PhRED) improved the thermostability.…”

Section: Introductionmentioning

confidence: 82%

“…10,11 Nevertheless, the poor thermostability of wild-type (WT) PhRED, which is also a common problem for all other beetle luciferases 23 and can affect the detection efficiency of in vivo BLI, 29 limits its application in demanding circumstances. Some attempts have been made to improve firefly luciferase thermostability by mutagenesis (e.g., Ppy, [30][31][32] Lcr, 33 Luciola lateralis (Lla), 34 Hotaria parvula (Hpa) 35 ). Among these, mutations of T217 (Lcr) and A217 (Lla) (i.e., I212 of PhRED) to the three most hydrophobic residues (I, V, and L) produced mutants with greater thermostability.…”

Section: Introductionmentioning

confidence: 99%

Enhanced red‐emitting railroad worm luciferase for bioassays and bioimaging

Nakajima

Niwa

et al. 2009

Protein Science

View full text Add to dashboard Cite

A luciferase from the railroad worm (Phrixothrix hirtus) is the only red-emitting bioluminescent enzyme in nature that is advantageous in multicolor luciferase assays and in bioluminescence imaging (BLI). However, it is not used widely in scientific or industrial applications because of its low activity and stability. By using site-directed mutagenesis, we produced redemitting mutants with higher activity and better stability. Compared with the wild-type (WT), the luminescent activities from extracts of cultured mammalian cells expressing mutant luciferase were 9.8-fold in I212L/N351K, 8.4-fold in I212L, and 7.8-fold in I212L/S463R; and the cell-based activities were 3.6-fold in I212L/N351K and 3.4-fold in N351K. The remaining activities after incubation at 37°C for 10 min were 50.0% for I212L/S463R, 31.8% for I212L, and 23.0% for I212L/ N351K, but only 5.2% for WT. To demonstrate an application of I212L/N351K, cell-based BLI was performed, and the luminescence signal was 3.6-fold higher than in WT. These results indicate that the mutants might improve the practicability of this signaling in bioassays and BLI.

show abstract

Development of a thermostable firefly luciferase

Cited by 57 publications

References 19 publications

Construction and Validation of Improved Triple Fusion Reporter Gene Vectors for Molecular Imaging of Living Subjects

Construction and Validation of Improved Triple Fusion Reporter Gene Vectors for Molecular Imaging of Living Subjects

Thermostabilization of Firefly Luciferases Using Genetic Engineering

Enhanced red‐emitting railroad worm luciferase for bioassays and bioimaging

Contact Info

Product

Resources

About