Isolated luciferases and uses thereofThe application is a divisional application, the application date of the original application is 11/22/2001, the application number is 01819433.8, and the invention name is 'isolated luciferase and application thereof'.
Technical Field
The invention relates to nucleotide and amino acid sequences of luciferases LuAL, Lu164, Lu16, Lu39, Lu45, Lu52 and Lu22, and activity and application thereof.
Technical Field
Luciferase enzyme
Luminescence means the emission of photons in the visible spectral range, which emission is generated by excitation of luminescent molecules. Unlike fluorescence, the energy of this light is not provided externally in the form of shorter wavelength radiation.
There is a difference between chemiluminescence and bioluminescence. Chemiluminescence refers to a chemical reaction that results in excitation of a molecule, which itself emits light when the excited electrons return to the ground state. If the reaction is catalyzed by an enzyme it is called bioluminescence. The enzyme involved in the reaction is generally referred to as luciferase.
For a review of luminogens, see Hastings et al 1995.
The luciferase is a peroxidase or a monooxygenase and a dioxygenase. The enzyme substrate that forms the starting material for the luminescent product is called fluorescein. Fluorescein varies from species to species. The quantum yields of the system are between 0.1 and 0.9 photons per conversion substrate molecule (Idelgaufts, 1993).
Luciferases can be classified according to their origin or enzymatic properties. A summary of certain types of luciferases is provided below:
bacterial luciferases
| Gene product | Biological organisms | Substrate | λ | Expression of | References/patents |
| Lux gene | Vibrio fischerii | FMN, dodecyl aldehyde NADH | 495nm | Cytoplasm of cells | Apley et al.，1985Gustafson G.，US5196524 |
Table 1: bacterial luciferases
Coel enterazine-dependent eukaryotic luciferase
| Gene product | Biological organisms | Substrate | λ | Expression of | References/patents |
| Renilla luciferase | Renillareniformis | Coelenterazine | 480nm | Cytoplasm of cells | Mathews et al.，1977Lorenz et al，1991Lorenz et al.1996Alan，P；WO0020619Milton J.，US5418155Roelant C.，WO9938999 |
| Vargula/Cypridia luciferases | Vargulahilgendorferii | Vargula fluorescein | 460nm | Secretion of | Thomspon et al.，1989Thompson et al.，1990Tora，JP05064583Tora，JP08027200Renard et al.，WO9520653 |
| Watasemia luciferase | Wataseniascintillans | Watasemia fluorescein | ？ | Cytoplasm of cells | Inoue et al.，1976 |
| Olophorus luciferase | Olophorusgracilirostris | Coelenterazine | 454 | Secretion of | Inouye et al，2000 |
| Aequorin | Aequoriaaequoria | Coelenterazine(CaActivated) | 470nm | Cytoplasm of cells | Headet al.2000Shimomura et al.，2000Jones et al.，1999Kendall et al.，1998Inouye et al.，1985Shimomura et al.，1969Cormier et al.，US5798441Cormier et al.，US5422266 |
| Obelin | Obelia | Coelenterazine | 470nm | Cytoplasm of cells | Matveev et al.，1999Berestovskaya，1999 |
Table 2: coelenterazine-dependent eukaryotic luciferases
Coe l ent eraz i ne-independent eukaryotic luciferase
| Gene product | Biological organisms | Substrate | λ | Expression of | Reference to the literature |
| Firefly luciferase | Photinuspyralis | Firefly luciferin, ATP | 550nm | Cytoplasm of cells | Webster et al.，1980Gould et al.，1988Sala-Newby et al，1992Bonin et al.，1994SherfB.，US5670356KIKK，JP09187281 |
Table 3: coelenterazine-independent eukaryotic luciferase
Various luciferases can also be distinguished based on the substrate specificity of some luciferases. The most important substrates include Coelenterazine (Jones et al 1999) and fluorescein, as well as derivatives of both substances. A schematic of these two substrates and their conversion by luciferase is shown below:
luciferase substrates
Some luciferase substrates are shown below by way of exampleAnd transformation thereof. All substrates shown herein are enzymatically converted with the release of light and carbon dioxide (CO)2) And consumes oxygen (O)2). The dependence of the reaction on a cofactor or energy carrier (e.g., ATP for firefly luciferase) is enzyme specific.
Coelenterazine
Aequorin emits light at a wavelength of 470 nm when it converts Coelenterazine into Coelenteramide.
Fluorescein
Fluorescein Hydroxyfluorescein
Firefly luciferin, when converted to hydroxyluciferin, emits light at a wavelength of 560 nanometers.
Vargula-fluorescein
Vargula-fluorescein Vargula-hydroxyfluorescein
Vargula luciferase emits light at a wavelength of 460 nm when converting Vargula luciferin to Vargula-hydroxyluciferin.
Reporter system
A reporter gene or indicator gene is generally a gene whose gene product can be readily detected by simple biochemical or histochemical methods. At least two types of reporter genes are distinguishable.
1. A resistance gene. A resistance gene is a gene whose expression causes a cell to develop resistance to an antibiotic or other substance. In the absence of this resistance gene, the presence of the antibiotic or other substance in the growth medium can result in cell death.
2. A reporter gene. In genetic engineering techniques, the product of a reporter gene is used as a fused or unfused indicator. The most common reporter genes include beta-galactosidase (Alam et al 1990), alkaline phosphatase (Yang et al 1997; Cullen et al 1992), luciferase and other photoproteins (Shinomura, 1985; PhillipsGN, 1997; Snowdowne et al 1984).
Classification of Metridia longa species
Arthropoda → Crustacea → Petalaria
Metridia longa belongs to the crustaceans, in particular to the copepoda or zooplankton.
Disclosure of Invention
Isolation of cDNA
To study the bioluminescent activity of Metridia longa species, samples were obtained from the white sea (biologicsche Station Kartesh, russia) and stored in liquid nitrogen. To prepare a cDNA library of Metridia longa, RNA was isolated by the method of Krieg (Krieg et al 1996) using isothiocyanate.
RT-PCR was performed to prepare cDNA. For this purpose, 10. mu.g of RNA were incubated with reverse transcriptase (Supersacrbt Gold II) according to the following protocol:
PCR 1.30 sec 95 deg.C
68 ℃ for 2.6 min
95 ℃ for 3.10 seconds
68 ℃ for 4.6 min
Step 4 was performed after step 3, and 17 cycles were performed
To inactivate the polymerase, the reaction product was incubated with proteinase K at 37 ℃ for 30 minutes and the cDNA was precipitated with ethanol. The cDNA was dissolved in water and incubated with SfiI at 37 ℃ for 1 hour. The reaction product was subjected to gel filtration to separate small fragments. The isolated cDNA was then ligated into SfiI cleaved and dephosphorylated lambda TriplEx2 vector. Then, to prepare a lambda phage expression library, the cloned cDNA fragment was encapsulated into lambda phage using SMART cDNA library construction kit (Clontech) in an in vitro packaging system.
Recombinant phages containing cDNA inserts with the potential to express colenterazine-dependent luciferase were identified by performing library screening.
For this purpose, the lawn obtained from E.coli XL1-Blue was spread on 90 mm dishes and incubated at 37 ℃ for 10 hours. Each dish was then infected with 2500 phages and then incubated at 37 ℃ for 8 hours to form plaques. The plates were then stored at 4 ℃ for 1 hour to harden the soft agarose.
For replica plating, the nitrocellulose membrane was saturated with E.coli XL1-Blue suspension and dried. The dried membrane was placed on the plaques for 60 seconds and then spread on a new agarose plate. The agarose plates were then incubated at 37 ℃ for 2 hours and 4 ml of SOB medium (+10mM MgSO. RTM. MgSO. RTM. medium) was added40.2% maltose). The lawn was isolated, resuspended in LB medium (+20mM IPTG), and cultured at 37 ℃ for 1 hour. The bacteria are harvested by centrifugation and disrupted by ultrasonication inAfter addition of Coelenterazin, the bioluminescent activity was measured in a luminometer.
The culture dish producing a positive bioluminescent signal is divided into sectors and a new replica plating is performed. Replica plating was continued until active individual plaques were identified. For subcloning of phage cDNA inserts of positive plaques, the procedure of the manufacturer of the SMART cDNA library construction kit was followed in pTriplEx2 vector in E.coli BM 25.8. pTriplEx2-cDNA transfected E.coli was cultured overnight at 37 ℃ in LB medium containing ampicillin at a concentration of 100. mu.g/ml. To obtain overexpression, the overnight cultures were diluted 1: 150 fold with LB medium and incubated for 1 hour at 37 ℃. Then, the culture was carried out by adding IPTG (isopropyl thiogalactoside) to a concentration of 20 mM. The induced medium was cultured at 37 ℃ for 1 hour, and the bacteria were harvested by centrifugation. The cells were disrupted by sonication in 0.5 ml of SM buffer. After addition of 10. mu.l of Coelenterazin (10)-4M, dissolved in methanol) followed by chemiluminescence measurement in a luminometer.
Three luciferases having Coelenterazine dependent luciferase activity were identified. The luciferases are named Lu164, LuAL and Lu 22. The luciferase will be disclosed in detail below.
The invention also relates to functional equivalents of these three luciferases. Functional equivalents are luciferases with a comparable substrate range, which are secreted and have at least 70% homology. Preferably 80% or 90% homology. Particular preference is given to a homology of 95%.
Luciferases are suitable as reporter genes for cell systems, in particular as receptors, ion channels, transfer vectors, transcription factors or induction systems.
Luciferases can be used in bacterial systems, for example, for titer determination or as substrates for biochemical systems, particularly proteases.
Luciferase may also be used as a reporter linked to antibodies or other proteins, e.g., for ELISA, for immunohistochemistry or for Western blotting.
Luciferase may be used in a BRET (bioluminescence resonance energy transfer) system.
Luciferases are also suitable for use as fusion proteins, for performing confocal microscopy assays, or for assaying protein-protein interactions.
Luciferase may also be used as a reporter linked to biotin, NHS, CN-Br or other binding mediators, e.g., for ELISA or for immobilization.
In addition, luciferase may be used as a reporter enzyme linked to a DNA or RNA oligonucleotide, e.g., for Northern and Southern blots or for real-time PCR.
The invention also relates to the purification of luciferase as a wild-type or marker protein and to the use of luciferase in an in vitro translation system.
Nucleotide and amino acid sequences
LuAL
The luciferase LuAL is a protein with a molecular weight of 23.7kDa and an isoelectric point of 8.32. The coding nucleotide sequence is as follows:
5`atggatatgagggttatctttgctcttgttttctcatcattggttcaggccaaatcaactgaattcgatccta
acattaacattgttggtttagaaggaaaatttggtataacaaaccttgagacggatttattcacaatatgggaga
caatggatgtcatcaaatcagatattacagatactgatagagtcagcaactttgttgcaactgaaaccgatgcta
accgtgggaaaatgcctggcaaaaaactgccactggcagttatcatggaaatggaagccaatgctttcaaagctg
gctgcaccaggggatgccttatctgtctttcaaaaataaagtgtacagccaaaatgaaggtgtacattccaggaa
gatgtcatgattatggtggtgacaagaaaactggacaggcaggaatagttggtgcaattgttgacattcccgaaa
tctctggatttaaggagatggcacccatggaacagttcattgctcaagttgatctttgcgctacctgcactactg
gatgtctcaaaggtcttgccaatgttaagtgctctgaactcctgaagaaatggctgcctggcagatgtgcaagtt
ttgctgacaagattcaaaaagaagttcacaatatcaaaggcatggctggagatcgttga3`
it can generate the following amino acid sequence:
MDMRVIFALVFSSLVQAKSTEFDPNINIVGLEGKFGITNLETDLFTIWETMDVIKSDITDTD
RVSNFVATETDANRGKMPGKKLPLAVIMEMEANAFKAGCTRGCLICLSKIKCTAKMKVYIPG
RCHDYGGDKKTGQAGIVGAIVDIPEISGFKEMAPMEQFIAQVDLCATCTTGCLKGLANVKCS
ELLKKWLPGRCASFADKIQKEVHNIKGMAGDR
and the following amino acid composition:
Ala：18(8.3％)Cys：10(4.6％)Asp：14(6.4％)Glu：12(5.5％)
Phe：10(4.6％)Gly：19(8.7％)His：2(0.9％)Ile：18(8.3％)
Lys：21(9.6％)Leu：15(6.9％)Met：10(4.6％)Asn：8(3.7％)
Pro：7(3.2％)Gln：5(2.3％)Arg：7(3.2％)Ser：9(4.1％)
Thr：15(6.9％)Val：14(6.4％)Trp：2(0.9％)Tyr：2(0.9％)
Lu164
the luciferase Lu164 is a protein with a molecular weight of 23.8kDa and an isoelectric point of 7.81. The coding nucleotide sequence is as follows:
5`atggatataaaggttgtctttactcttgttttctcagcattggttcaggcaaaatcaactgaattcgatccta
acattgacattgttggtttagaaggaaaatttggtataacaaaccttgagacggatttattcacaatatgggaga
caatggaggtcatgatcaaagcagatattgcagatactgatagagccagcaactttgttgcaactgaaaccgatg
ctaaccgtggaaaaatgcctggcaaaaaactgccactggcagttatcatggaaatggaagccaatgctttcaaag
ctggctgcaccaggggatgccttatctgtctttcaaaaataaagtgtacagccaaaatgaaggtgtacattccag
gaagatgtcatgattatggtggtgacaagaaaactggacaggcaggaatagttggtgcaattgttgacattcccg
aaatctctggatttaaggagatggcacccatggaacagttcattgctcaagttgaacgttgcgcttcctgcacta
ctggatgtctcaaaggtcttgccaatgttaagtgctctgaactcctgaagaaatggctgcctgacagatgtgcaa
gttttgctgacaagattcaaaaagaagttcacaatatcaaaggcatggctggagatcgttga3`
it can generate the following amino acid sequence:
MDIKVVFTLVFSALVQAKSTEFDPNIDIVGLEGKFGITNLETDLFTIWETMEVMIKADIADT
DRASNFVATETDANRGKMPGKKLPLAVIMEMEANAFKAGCTRGCLICLSKIKCTAKMKVYIP
GRCHDYGGDKKTGQAGIVGAIVDIPEISGFKEMAPMEQFIAQVDRCASCTTGCLKGLANVKC
SELLKKWLPDRCASFADKIQKEVHNIKGMAGDR
and has the following amino acid composition:
Ala：21(9.6％)Cys：10(4.6％)Asp：15(6.8％)Glu：13(5.9％)
Phe：10(4.6％)Gly：18(8.2％)His：2(0.9％)Ile：18(8.2％)
Lys：22(10.0％)Leu：14(6.4％)Met：10(4.6％)Asn：7(3.2％)
Pro：7(3.2％)Gln：5(2.3％)Arg：7(3.2％)Ser：8(3.7％)
Thr：14(6.4％)Val：14(6.4％)Trp：2(0.9％)Tyr：2(0.9％)
Lu22
the luciferase Lu22 is a protein with a molecular weight of 20.2kDa and an isoelectric point of 7.89. The coding nucleotide sequence is as follows:
5`atgggagtcaaacttatttttgctgttgtttgtgtcgcagttgcccaggctgccacaattcaggaaaattttg
aagacattgatcttgtagccataggtggcagctttgcatcagatgttgatgctaacagaggtggacatggtggac
atcctggcaaaaagatgccaaaagaagtacttatggaaatggaagccaatgctaaacgagctggctgccacaggg
gttgtctggtttgtctgtcacacatcaagtgcacagcacaaatgcagaagtttatcccaggaagatgccatagtt
atgcaggagacaaggattctgctcagggaggaattgccggtggtgccattgttgatatacctgaaattgccggat
ttaaagaaatgaagcccatggaacagttcattgctcaagttgatctctgtgaagattgcacaactggatgcctca
aaggtcttgccaatgttcattgctctgatctcctgaagaagtggctgccatcaagatgtaagacatttgcttcca
aaattcaatctcaagtggataccatcaaaggtttggctggagatcgttga3`
it can generate the following amino acid sequence:
MGVKLIFAVVCVAVAQAATIQENFEDIDLVAIGGSFASDVDANRGGHGGHPGKKMPKEVLME
MEANAKRAGCHRGCLVCLSHIKCTAQMQKFIPGRCHSYAGDKDSAQGGIAGGAIVDIPEIAG
FKEMKPMEQFIAQVDLCEDCTTGCLKGLANVHCSDLLKKWLPSRCKTFASKIQSQVDTIKGL
AGDR
and has the following amino acid composition:
Ala：21(11.1％)Cys：11(5.8％)Asp：12(6.3％)Glu：9(4.7％)
Phe：7(3.7％)Gly：21(11.1％)His：6(3.2％)Ile：13(6.8％)
Lys：16(8.4％)Leu：12(6.3％)Met：7(3.7％)Asn：4(2.1％)
Pro：6(3.2％)Gln：9(4.7％)Arg：6(3.2％)Ser：9(4.7％)
Thr：6(3.2％)Val：13(6.8％)Trp：1(0.5％)Tyr：1(0.5％)
the above sequences are also shown in the sequence listing.
Enzymatic activity and biochemical characterization of luciferase
The proteins LuAL, Lu164 and Lu22 are enzymes that emit light when transformed into Coelenterazin. Therefore, they belong to luciferases. Luciferase can be efficiently expressed in bacteria and eukaryotic cells. The luciferases LuAl, Lu164 and Lu22 expressed in the eukaryotic cells are secreted, and the secretion related to the bacterial expression does not occur.
The activity of luciferase is temperature dependent. The optimal temperatures for determining the luciferases LuAL and Lu164 are 22 deg.C (LuAL) and 27 deg.C (Lu164), respectively. The optimal temperature for luciferase Lu22 activity is 4 ℃ or less.
Detailed Description
Examples
plasmid/Structure
Vectors used for preparing the structures disclosed hereinafter are the vectors pcDNA3.1(+) and pTriplEx2, which are available from Clontech, and the vector pASMplr (with the inherent structure of a cAMP-sensitive promoter element; cre). The derivatives of the vectors were named pcDNA3-x, pTriplEx2-x and pASM-x.
LuAL
FIG. 1 shows plasmid maps of the vectors pTriplEX2-LuAL, pcDNA3-LuAL and pASM-LuAL.
FIG. 2 shows plasmid maps of the vectors pTriplEX2-Lu164, pcDNA3-Lu164 and pASM-Lu 164.
FIG. 3 shows plasmid maps of the vectors pTr iplEX2-Lu22, pcDNA3-Lu22 and pASM-Lu 22.
Coelenterazin derivatives as Lu164 substrates
To identify the substrate for Lu164, 10. mu.l of a different Coelenterazin derivative (10) were used in each case-4M) was incubated with 10. mu.l of supernatant from the CHO-pcDNA3-Lu164 cell line and luminescence was determined. Coelenterazine was obtained from Molecular Probes (USA).
The luciferases LuAL and Lu22 have no difference compared with the luciferase Lu 164. Unmodified Colenterazin (panel B, Coelenterazina) was identified as the best substrate for Lu164, LuAl and Lu 22.
FIG. 4 shows the Coelenterazin derivative as a potential substrate for Lu164 and the curve obtained by measuring the luminescence in a luminometer (RLU, relative optical units) for 30 seconds at a voltage of 8.7 kV; and a molecular structure schematic diagram of the Coelenterazin derivative.
The luciferases Lu164, LuAL and Lu22 depend on Coelenterazin for the enzymatically active bacterial expression
Bacterial expression was carried out in E.coli strain BL21(DE3) by transforming the bacteria with the expression plasmids pTriplEX2-Lu164, pTriplEX2-LuAL and pTriplEX2-Lu 22. The transformed bacteria were cultured in LB medium at 37 ℃ for 3 hours, and expression was induced by adding IPTG to a final concentration of 1mM for 4 hours. Induced bacteria were harvested by centrifugation, resuspended in PBS, and disrupted by sonication. Coelenterazin (10)-4M, dissolved in methanol) or fluorescein (firefly fluorescein) was added to 5 microliters of lysate (5mg/ml) and chemiluminescence was measured.
The measurement was carried out at a voltage of 9.5kV for 30 seconds in terms of RLU (relative luminescence units). The values determined for Lu164, LuAL and Lu22 were 230000, 320000 and 260000RLU, respectively. The expression was carried out in E.coli BL21(DE3) using the vectors pTriplEx2-Lu164, pTriplEx2-LuAL and pTriplEx2-Lu 22.
Eukaryotic expression
In transient experiments, constitutive eukaryotic expression in CHO cells was affected by transfecting the cells with expression plasmids pcDNA3-Lu164, pcDNA3-LuAL and pcDNA3-Lu 22. For this purpose, 10000 cells suspended in DMEM-F12 medium were seeded in each well of a 96-well microtiter plate and cultured overnight at 37 ℃. Transfection was performed using the Fugene6 kit (Roche) according to the manufacturer's instructions. The transfected cells were cultured overnight at 37 ℃ in DMEM-F12 medium. Adding Coelenterazin (10)-4M, dissolved in methanol), chemiluminescence was measured in the medium (5 μ l) and cell lysate (5 μ l) at 9.5kV for 30 seconds at room temperature with a luminometer.
The values determined for Lu164, LuAL and Lu22 were 680000, 670000 and 510000RLU (relative light units), respectively. The expression was carried out in CHO cells using the vectors pcDNA3-Lu164, pcDNA3-LuAL and pcDNA3-Lu 22.
Luminescence spectra of luciferases Lu164, LuAL and Lu22
For the determination of the luminescence spectra, E.coli BL21(DE3) cells were transformed with the plasmids pTriplEx2-Lu164, pTriplEx2-LuAL and pTriplEx2-Lu22 and overexpressed as described in section 3.1 below. 50 microliters of Coelenterazin (10) was added-4M) was added to a 100 microliter volume of bacterial lysate and the luminescence spectrum was determined. The luminescence spectrum curve of luciferase is shown below.
For the luciferases LuAL, Lu164 and Lu22, the maximum luminescence generated by said substrate conversion occurs at a wavelength of about 490 nm.
FIG. 5 shows luminescence spectra (RLU, relative luminescence units) of luciferases Lu164(A), LuAL (B) and Lu22(C) after bacterial expression.
Secretion of Lu164, LuAL and Lu22 luciferases from CHO cells, Lu164 and LuAL as examples
To characterize the expression of the luciferases LuAL, Lu164 and Lu22 in eukaryotic cells, CHO cells were stably transfected with plasmids pcDNA3-LuAl, pcDNA3-Lu164, pcDNA3-Fireluc and pcDNA3.1 (+). The resulting clones were cultured in DMEM-F12 medium. Firefly luciferase was used as a positive control for non-secreted luciferase. The potential endogenous activity in the CHO parent cell was tested using the plasmid pcDNA3.1(+) as a control plasmid.
To detect luciferase secretion, 2000 cells were plated onto 384-well microtiter plates. After 24 hours, the medium was removed and the cells were washed with Tyrode solution and 30 microliters of fresh medium was added. For firefly luciferase, 5. mu.l of Coelenterazin (10) was added-4M) or fluorescein, first measured (0 hours) with a luminometer at a voltage of 9.5kV for 30 seconds. The measurement is carried out for 1 to 5 hours after the lapse of 1 to 5 hours.
FIG. 6 shows the increase of luciferase activity in the medium with time. Firefly luciferase is not secreted. The luciferases LuAL, Lu164 and Lu22 are secreted luciferases.
FIG. 6 shows luciferase activity (RLU, relative luminescence units; h is h; Firefly: Firefly luciferase) in CHO cell culture medium (5. mu.l) after transfection of cDNA-free inserts with pcDNA3-LuAL, pcDNA3-Firefly, pcDNA3-Lu164 and pcDNA3 as control vectors.
Dependence of luciferase activity on temperature
To determine the temperature dependence of the luciferases Lu22, Lu164 and LuAL, CHO cells were transiently transfected with the vectors pcDNA3-Lu22, pcDNA3-Lu164 and pcDNA3-LuAL, and luciferase activity in the supernatant was determined at 0-47 ℃. For this purpose, the cell supernatants and the Coelenterazin solutions were allowed to acclimate to the assay temperature for 5 minutes. The measurement was carried out in a luminometer at a voltage of 9.5kV for 30 seconds.
FIG. 7 shows the luminescence of the luciferases LuAl, Lu164 and Lu22 determined depending on the temperature. The optimal temperature for luciferase activity for LuAL is 27 ℃. The optimum temperature was 22 ℃ for Lu164 and 4 ℃ or less for Lu 22.
FIG. 7 shows temperature-dependent luciferase activity (RLU: relative luminescence units; medium: DMEM-F12+ 10% FCS) in CHO cell culture medium (5. mu.l) after transfection with pcDNA3-LuAL, pcDNA3-Firefly and pcDNA3-Lu164
Induction of the expression of the luciferases Lu164, LuAL and Lu22 in CHO cells, taking LuAL as an example
In transient experiments, eukaryotic expression was induced in CHO cells by transfecting the cells with the expression plasmid pASM-LuAL. For this purpose, 10000 cells suspended in DMEM-F12 medium were seeded in each well of a 96-well microtiter plate and cultured overnight at 37 ℃. With Fugene
6 kit (Roche) transfection was performed according to the manufacturer's instructions. The transfected cells were cultured overnight at 37 ℃ in DMEM-F12 medium. Then using Forskolin (Forskolin) (10)-5M) inducing the cells for 5 hours. Adding Coelenterazin (10)-4M, dissolved in methanol), the chemiluminescence of the medium and cell lysate was measured with a luminometer at a voltage of 9.5kV for 30 seconds.
FIG. 8 shows induced expression of LuAL in CHO cells. Expression was induced with forskolin (10-5M) at 37 ℃ for 5 hours. Determination of the Activity in 10 microliter cell supernatants (RLU: relative luminescence units; Induction factor: ratio of induced to uninduced RLU) luciferase Lu164, LuAL and Lu22 were used as reporter genes in the cell System, exemplified by the reporters NPY2 and A2A, and LuAL was used as reporter gene
To be able to analyze the activation of the G protein binding receptor by receptor-specific ligands in a cell-based system, the cDNA sequence of the luciferase LuAL was cloned into the expression vector pASMplr. The expression vector pASMplr contains a cAMP-sensitive promoter element (CRE), which is capable of indirectly determining the intracellular concentration of cAMP. In this system, luciferase is used as a reporter gene.
The use of the luciferases Lu22, Lu164 and Lu22 as reporter genes in cell systems is illustrated by the G protein binding receptors NPY2 (neuropeptide receptor 2) and A2A (adenosine receptor 2 a). For this purpose, the stable clone CHO-pASM-LuAL was transiently transfected with the vector pcDNA3-NPY2 or pcDNA 3-A2A. The receptor NPY2 is a Gi-binding receptor, while the A2A receptor is a Gs-binding receptor.
The A2A receptor was activated by addition of 1 μ M NECA for 4 hours. In the presence of 10-5NPY2 receptors were activated by addition of 10 μ M NPY2 peptide under conditions of M forskolin. Adding Coelenterazin (10)-4M) followed by measurement of luciferase activity in the medium (30. mu.l) with a voltage of 9.5kV using a luminometer for 30 seconds.
FIG. 9 shows the use of luciferase as a reporter gene for cell systems, exemplified by the G protein binding receptors A2A and NPY2 (RLU: relative light units).
Drawings
FIG. 1: plasmid maps of the vectors pTriplex2-LuAL, pcDNA3-LuAL and pASM-LuAL.
FIG. 2: plasmid maps of the vectors pTriplEX2-Lu164, pcDNA3-Lu164 and pASM-Lu 164.
FIG. 3: plasmid maps of the vectors pTriplEX2-Lu22, pcDNA3-Lu22 and pASM-Lu 22.
FIG. 4: a Coelenterazin derivative as a potential substrate for Lu 164.
A. The curve obtained by measuring the luminescence in a luminometer (RLU, relative optical units) at a voltage of 8.7kV for 30 seconds;
schematic molecular structure of coelenterazin derivatives.
FIG. 5: luminescence spectra (RLU, relative luminescence units) of luciferases Lu164(a), lual (b), and Lu22(C) after bacterial expression.
FIG. 6: luciferase activity (RLU, relative luminescence units; h is h; Firefly: Firefly luciferase) in CHO cell culture medium (5. mu.l) after transfection of the cDNA-free insert with pcDNA3-LuAL, pcDNA3-Firefly, pcDNA3-Lu164 and pcDNA3 as control vectors.
FIG. 7: temperature-dependent luciferase activity in CHO cell culture medium (5. mu.l) after transfection with pcDNA3-LuAL, pcDNA3-Firefly and pcDNA3-Lu164 (RLU: relative luminescence units; medium: DMEM-F12+ 10% FCS).
FIG. 8: inducible expression of LuAL in CHO cells. Expression was induced with forskolin (10-5M) at 37 ℃ for 5 hours. The activity (RLU: relative luminescence units; induction factor: ratio of induced to non-induced RLU) was determined in 10. mu.l cell supernatants.
FIG. 9: the use of luciferase as a reporter gene for cell systems, exemplified by the G protein binding receptors A2A and NPY2 (RLU: relative light units).
Reference/patent document
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