Higher intensity luciferase

Akaluc luciferase providing brighter and red-shifted luminescence

Firefly bioluminescence systems have been commonly used as a imaging tool of biological phenomena. However, it have not been strong enough for imaging signals in deep tissues, because of low permeabilization of substrates. Recently, Dr. Atsushi Miyawaki and Dr. Satoshi Iwano of the RIKEN Center for Brain Science, and Dr. Shojiro Maki of the University of Electro-Communications developed a system of artificial bioluminescence AkaBLI that enables noninvasive signal observation in deep tissue of living animals.

The AkaBLI system consists of an artificial substrate AkaLumine with improved tissue permeability and an artificial luciferase Akaluc optimized to AkaLumine. The intensity of the luminescence of AkaBLI system is 100 to 1000 folds brighter than the conventional systems.
The expression vector of artificial luciferase Akaluc has been deposited by Dr. Miyawaki’s lab and it is now available from the DNA Bank.

Reference
Nakashiba, T. et al., Development of two mouse strains conditionally expressing bright luciferases with distinct emission spectra as new tools for in vivo imaging. Lab. Anim. (NY). 2023 Sep 7. doi: 10.1038/s41684-023-01238-6. Epub ahead of print. PMID 37679611.
Iwano, S. et al., Single-cell bioluminescence imaging of deep tissue in freely moving animals. Science 359 (6378): 935-939 (2018). PMID 29472486.
Press Release and Blog
Development of two mouse strains conditionally expressing bright luciferases with distinct emission spectra as new tools for in vivo imaging. (2023/9/8, RIKEN BRC)
In living color: seeing cells from outside the body with synthetic bioluminescence (2018/2/23, RIKEN)
In living color: imaging the brain with synthetic bioluminescence (2018/2/23, It Ain’t Magic Blog)
DNA resource
pcDNA3 Venus-Akaluc (cat# RDB15781)
pAAV2 SynTetOff Venus-Akaluc (cat# RDB15782)
pAAV2 TRE Venus-Akaluc (cat# RDB15783)

(2018.05.07 N.N.)

Brighter than commonly used luciferase!

These luciferases have at most four-time maximum luminous intensity than that of widely used luciferase of the Photinus pyralis, a common North American firefly. These luciferases as a reporter, allow to monitor gene expression with high sensitivity and time-lapse observation.


Observation of luminescence of transfected HeLa cells. HeLa cells were transfected with expression vectors harbouring luciferase coding regions, respectively. Luminescence was observed under the condition of 2 mM D-luciferin. Left, PmatLuc1, DkumLuc1, MALuci2Luc. Right, SflaRE1Luc, PsagRE1Luc.

Catalog no.	Name of clone	Origin	Intracellular luminescence intensity(vie, Phothinus pyralis)	Maximal absorption wavelength (pH8, in vitro)		misc.
Catalog no.	Name of clone	Origin		25℃	37℃	misc.
RDB14359	pPmatLuc1	Pyrocoelia matsumurai	4 times or more	560 nm	567 nm	luciferase cDNA is cloned in pUC19 vector.
RDB14360	pDkumLuc1	Drilaster kumejimensis	4 times	558 nm	562 nm
RDB14363	pMALuci2Luc	Malaysian Luciola sp. (1)	2 times	556 nm	557 nm
RDB14362	pSflaRE1Luc	Stenocladius flavipennis	2 times	608 nm	609 nm
RDB14361	pPsagRE1Luc	Pristolycus sagulatus	2 times	605 nm	606 nm

¹The luciferase gene originated from Malaysian firefly was developed together with Perak State Development Corporation and Nimura Genetic Solutions in conformity with the Convention on Biological Diversity.

Multiple colors with one substrate!

Green-, Yellow- or Red-emission can be obtained by using D-luciferin as a substrate of each luciferase. Simultaneous monitoring of two genes can be allowed by using green- and red-luciferases.

High stabilities under various pH conditions!

These luciferases are less susceptible under variety of pH or temperature and the stable results can be obtained. Please visit our web site to see results of luminescence at 25℃ and 37℃.

Purified recombinant luciferases were mixed with GTA Buffer (final conc. 200 mM), ATP (final conc. 2 mM), MgSO₄ (final conc. 2 mM), D-Luciferin (final conc. 1 mM) and luminescence under the different pH conditions were observed. Left, 25℃. Right, 37℃.
PmatLuc1
DkumLuc1
MALuci2Luc
PsagRE1Luc
SflaRE1Luc

Reference
Nakashiba, T. et al., Development of two mouse strains conditionally expressing bright luciferases with distinct emission spectra as new tools for in vivo imaging. Lab. Anim. (NY). 2023 Sep 7. doi: 10.1038/s41684-023-01238-6. Epub ahead of print. PMID 37679611.
Akiyoshi R, Ogoh K, Suzuki H. 2012. Firefly luciferase. Japan patent unregistered 2012-235756
Akiyoshi R, Ogoh K, Suzuki H. 2013. Firefly luciferase. Japan patent unregistered 2013-74861
Ogoh K, Akiyoshi R, Suzuki H. 2013. Firefly luciferase. Japan patent unregistered 2013-138668
Ogoh K, Akiyoshi R, Suzuki H. 2013. Firefly luciferase. Japan patent unregistered 2013-81459
Akiyoshi R, Ogoh K, Suzuki H. 2015. FIREFLY LUCIFERASE, Patent publication US2015/0291938 A1
Press Release
Development of two mouse strains conditionally expressing bright luciferases with distinct emission spectra as new tools for in vivo imaging. (2023/9/8, RIKEN BRC)

Artificial Luciferase Variants

Catalog #	Name of clone	Running title	Publication
RDB19616	ALuc23	Expression vector of artificial luciferase variant ALuc23 derived from the copepod gene database for mammalian cell imaging.	Kim, S.B. et al., 2013
RDB19617	ALuc49	Expression vector of artificial luciferase variant ALuc49 derived from the copepod gene database for mammalian cell imaging.	Kim, S.B. et al., 2017

Reference

Kim, S.B. et al. (2013) Bioconjug. Chem. 24 (12): 2067-2075. PMID 24237362
Kim, S.B. et al. (2017) ACS Comb. Sci. 19 (9): 594-599. PMID 28742969

JNC Corporation’s Resource

Aequorea victoria Aequorin

Catalog #	Name of clone	Running title	Publication
RDB19836	pAQ-B	Expression vector of A. victoria Aequorin, wild type.	Inouye, S. et al., 1985
RDB19830	pJN-opAQ	Expression vector of A. victoria Aequorin, codon-optimized for human (opAQ).	Inouye, S. et al., 1985
RDB19847	pJNC-opAQ	Mammalian expression vector of A. victoria Aequorin, codon-optimized (opAQ).	Inouye, S. et al., 2015
RDB19848	pJNC-COsp-opAQ	Mammalian expression vector of human cytochrome C oxidase signal peptide sequence(COsp) and A. victoria Aequorin, codon-optimized (opAQ).	Inouye, S. et al., 2015

Clytia gregaria ClytinI and ClytinII

Catalog #	Name of clone	Running title	Publication
RDB19837	pCL41	Expression vector of C. gregaria ClytinI, wild type.	Inouye, S. et al., 1993
RDB19838	pCL31	Expression vector of C. gregaria ClytinII, wild type.	Inouye, S., 2008
RDB19831	pJN-opCLII	Expression vector of C. gregaria ClytinII, codon-optimized for human (opCLII).	Inouye, S. et al., 2015
RDB19849	pJNC-opCLII	Mammalian expression vector of C. gregaria ClytinII, codon-optimized (opCLII).	Inouye, S. et al., 2015
RDB19850	pJNC-COsp-opCLII	Mammalian expression vector of human cytochrome C oxidase signal peptide sequence(COsp) and C. gregaria ClytinII, codon-optimized (opCLII).	Inouye, S. et al., 2015

Renilla reniformis luciferase

Catalog #	Name of clone	Running title	Publication
RDB19853	pJNC-opRL	Mammalian expression vector of R. reniformis luciferase, codon-optimized (opRLuc).	Inouye, S. et al., 2015
RDB19854	pJNC-opRL547	Mammalian expression vector of R. reniformis Red-shiffted luciferase, codon-optimized (opRLuc547), 547 nm peak.	Unpublished bioresource
RDB19832	pJN-RLBP	Expression vector of R. reniformis luciferin-binding protein (RLBP).	Inouye, S. et al., 2007

Luciola cruciata luciferase

Catalog #	Name of clone	Running title	Publication
RDB19833	pJN-opLcLuc	Expression vector of L. cruciata firefly luciferase, codon-optimized for human (opLcLuc).	Inouye, S. et al., 2015

Photinus pyralis luciferase

Catalog #	Name of clone	Running title	Publication
RDB19834	pJN-opPyLuc	Expression vector of P. pyralis firefly luciferase, codon-optimized for human (opPyLuc).	Inouye, S. et al., 2015

Oplophorus gracilorostris luciferase

Catalog #	Name of clone	Running title	Publication
RDB19840	pKAZ-412	Expression vector of O. gracilorostris 19 kDa Protein of Oplophorus Luciferase (KAZ), catalytic component.	Inouye, S. et al., 2000
RDB19841	pJN-eKAZ	Expression vector of O. gracilorostris enhanced 19 kDa Protein of Oplophorus Luciferase (eKAZ).	Inouye, S. et al., 2014
RDB19851	pJNC-eKAZ	Mammalian expression vector of O. gracilorostris enhanced 19 kDa Protein of Oplophorus Luciferase (eKAZ).	Inouye, S. et al., 2014
RDB19852	pJNC-GLsp-eKAZ	Mammalian expression vector of signal peptide sequence of G. princeps Gaussia luciferase (GLsp) and O. gracilorostris enhanced 19 kDa Protein of Oplophorus Luciferase (eKAZ).	Inouye, S. et al., 2014
RDB19842	pOL23	Expression vector of O. gracilorostris 35 kDa protein of Oplophorus luciferase, unknown function.	Inouye, S. et al., 2000

Gaussia princeps luciferase

Catalog #	Name of clone	Running title	Publication
RDB19845	pJNC-hINS-GLuc	Mammalian expression vector of human preproinsulin (hINS) and G. princeps Gaussia luciferase (GLuc).	Suzuki, T. et al., 2011a
RDB19846	pJNC-hMMP2-GLuc	Mammalian expression vector of human matrix metalloprotease-2 (hMMP2) and G. princeps Gaussia luciferase (GLuc).	Suzuki, T. et al., 2011b
RDB19855	pJNC-pGLuc	Mammalian expression vector of G. princeps Gaussia luciferase, codon-optimized (GLuc).	Inouye, S. et al., 2016

Vibrio fischeri NAD(P)H-flavin oxidoreductase

Catalog #	Name of clone	Running title	Publication
RDB19835	pJN-FRase	Expression vector of V. fischeri NAD(P)H-flavin oxidoreductase (FRase).	Inouye, S., 1994

Reference

Inouye, S. et al. (1985) Proc. Natl. Acad. Sci. U S A. 82 (10): 3154-3158. PMID 3858813
Inouye, S. et al. (1993) FEBS Lett. 315 (3): 343-346. PMID 8422928
Inouye, S. (1994) FEBS Lett. 347 (2-3): 163-168. PMID 8033996
Inouye, S. et al. (2000) FEBS Lett. 481 (1): 19-25. PMID 10984608
Inouye, S. et al. (2007) Protein Expr. Purif. 52: 66-73. PMID 16997571
Inouye, S. J. Biochem. 143 (5):711-717, 2008. PMID 18296715
Inouye, S. et al. (2014)Biochem. Biophys. Res. Commun. 445 (1): 157-162. PMID 24491536
Inouye, S. et al. (2015) Protein Expr. Purif. 109: 47-54. PMID 25665506
Inouye, S. et al. (2016) Protein Expr. Purif. 128: 93-100. PMID 27506135
Suzuki, T. et al. (2011a) Anal. Bichem. 415 (2): 182-189. PMID 21477579
Suzuki, T. et al. (2011b) Plos One. 6 (9): e25243. PMID 21969874

Bacterial luciferase

Vibrio harveyi luciferase

Catalog #	Name of clone	Running title	Publication
RDB18951	pHLUX20	Expression vector of bacterial luciferase in Nitrosomonas europaea as a host strain.	Iizumi, T., 1998; Iizumi, T., 1997

Reference

Iizumi, T. et al. (1998) Appl. Environ. Microbiol. 64 (10): 3656-3662. PMID 9758781
Iizumi, T. and Nakamura, K. (1997) Appl. Environ. Microbiol. 63 (5): 1777-1784. PMID 9143112

Arranged vector

Catalog no.	Name of resource	Type
RDB19350	pGL4.14-E1bTATA Luciferase reporter vector of the minimal adenovirus E1b promoter sequence.
RDB03988	pCMVluc+ Expression vector of firefly luciferase
RDB06411	pGL3-RSV Expression vector of firefly luciferase, RSV promoter
RDB03989	pCAluc+ Expression vector of firefly luciferase
RDB02443	pCAcc-Luc+ Expressing luciferase under the control of CAG promoter
RDB02444	pLRICALuc+ Shuttle vector to generate recombinant adenovirus
RDB02968	pTcf7wtluc Reporter plasmid carring TCF-binding consensus sequence
RDB02969	pTcf7mtluc Reporter plasmid carring TCF-binding consensus sequence
RDB07031	ptk-luc Reporter construct with luciferase expression under tk promoter
RDB07032	pFGF4tk-luc Reporter construct with luciferase expression under FGF4 and tk promoter
RDB02453	AxCA Luc+ Recombinant adenovirus expressing luciferase gene
RDB04026	pKM2L Reporter construct with Renilla luciferase gene
RDB05549	pKM2L-pvTK HSV herpes simplex virus thymidine kinase (HSV-TK)
RDB05550	pKM2L-pvSV40 SV40 simian virus 40 early enhancer/promoter region (SV40)
RDB05551	pKM2L-pvCMV CMV cytomegalovirus immediate enhancer/promoter (CMV)
RDB06408	pRL-CMV mut1 Expression vector of Renilla luciferase, mutated CMV promoter
RDB06409	pRL-CMV mut2 Expression vector of Renilla luciferase, mutated CMV promoter
RDB06410	pRL-RSV Expression vector of Renilla luciferase, RSV promoter

Miscellaneous

Promoter Collection: constructs deposited by individual researchers and constructed by the Gene Engineering Division.

(GRP0025e 2016.11.16 N.N.)

2023.09.28