Gene, 96 (1990) 249-255 Elsevier
249
GENE 03803
Photoprotein aequorin: use as a reporter enzyme in studying gene expression in mammalian cells (Bioluminescence; calcium ion; CAT assay; eukza,yotic vectors; mammalian geae promoters; recombinant DNA; transfection)
Hiroshi Tanahashi', Takashi Ito b, Satoshi Inouye c, Frederick I. Tsuji d,e and Yoshiyuki Sakaki a ° Research Laboratory for Genetic Information, Kyushu University, Fukuoka 812 (Japan); ~ Institute of Tropical Medicine, Nagasata" University, Nagasaki 852 (Japan) Te1.(0958)47-2111; c Chisso Corporation, Kamaridani-2, Yokohama 236 (Japan) Te1. (045) 701-2443 ; d Osaka Bioscience Institute, Suita, Osaka 565 (Japan) Tel. (06)872-481.~; and e Marine Biology Research Division, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA 92093 (U.S.A.) Tel. (619)534-4245 Received by A. Nakazawa: 9 April 1990 Revised: 23 May 1990
Accepted: 25 May 1990
SUMMARY
Aequorin is a luminescent protein present in the jellyfish Aequoria victoria which emits light (at 460 nm) in the presence of Ca 2 +. We report here that aequorin can be used as a reporter enzyme to monitor gene expression in eukaryotic cells. A cDNA encoding apoaequorin was fused to several eukaryotic !,romoters, including those of SV40, RSV and the HSV-I tk gene, and introduced into several cell lines such as CV-I, COS and HeLa. At appropriate times after transfection, the aequorin activities in cell extracts were measured by monitoring the intensity of fight emitted at 460 nm when triggered by Ca 2 + by the use of a photomultiplier photometer. The aequorin assay was shown to be as sensitive as the conventional CAT assay, and the relative activities of various promoters estimated by the aequorin assay were in general agreement with those obtained by the CAT assay. The aequorin assay c~m be done within 6-7 h from the preparation of extract to the measurement of activity without using radioactive compounds.
INTRODUCTION
Currently, the regulation of eukaryotic gene expression is one of the most important and attractive subjects of study in molecular biology. For the analysis of the regulatory Correspondenceto: Dr. Y. Sakaki, Research Laboratory for Genetic Information, Kyushu Univecsity 3-1-1 Maidashi, Higashi-ku, Fukuoka 812 (Japan) Tel. (092)641-1151 ext. 3461; Fax (092)632-2375. Abbreviations: aaeq, gene (DNA) encoding apoaequorin; aeq, gene (DNA) encoding aequorin; bp, base pair(s); Cm, chloramphenicol; CAT, Cm acetyltransferase; HSV-I, Herpes simplex virus type I; IU, international unit(s); LTR, long terminal repeat; MT-I, gene encoding methailothionein I; nt, nucleotide(s); PBS(-), Ca 2+- and Mg2+-free phosphate-buffered saline (8 g NaCI/0.2 g KCI/I.15 g Na2HPO4/0.02 g KH2PO4 per liter of distilled water); Pn, penicillin G potassium salt; rlu, relative light unit(s); RSV, Rous sarcoma virus; SV40, simian virus 40; tk, gene encoding thymidine kinase. 0378-1119/90/$03.50 © 1990 Elsevier SciencePublishers B.V. (BiomedicalDb,ision)
mechanism, an appropriate reporter gene is often fused to possible regulatory sequences, and the resultant fusion gene is introduced into eukaryotic cells to monitor the effects of the sequences on gene expression. Several reporter genes have been used for this purpose, s~lch a-~those for chloramphenicol acetyltransferase (CAT) (German et al., 1982), //-galactosidase (An et al., 1982), human growth h~rmone (Selden et al., 1986), firefly luciferase (de Wet et aL, 1987) and alkaline phosphatase (Berger et al., 1988; Henthorn et al., 1988; Yoon et al., 1988). We have previously isolated a full-length aeq cDNA, encoding aequorin, a luminescent protein which emits light with a peak at 460 nm in the presence of Ca 2 + (Inouye et al., 1985; Tsuji et al., 1986): aequorin 3 caz+ ~hv + CO2 + apoaequorin+ coelenteramide ~ _ [ coelenterazine+ EDTA +'~ I O2 + 2-mercaptoethanol _!
250
(k) PMI
Pstl
~
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I ~
.Kpnl
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n,,,u,,,
I "
~
pSV2AQ
| | Kpnl
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Barn H HI
Barn Hi I "~mmm"~ BamHI " His dill
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Barn HI Hlnd;ll Sphl Pstl Sail EcoR Kpnl K/
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pSV?gpt-dedved SV40 smsll-t Inlron end polysdenylstlon signal ~
LTR of RSV
,.-od:ng sequence olaaeacONA
~
MTJ promoter
non-ceding sequence of aaeacDNA
~
SV40early promoter
HSV-1 t k gene promoter
(C) Qpoaequorln N-terminus
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.
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251 Aequorin seems to have some advantages as a reporter enzyme. For example, the quantum yield of the reaction is reasonably high (0.15) (Shimomura and Johnson, 1970; Johnson and Shimomura, 1978), and the assay of aequorin can be done within several h from the preparation of cell extracts to the end of assay withota using radioactive compounds. The activity can be easily and quantitatively measured by using a conventional photomultiplier photometer. Thus, a unique system can be developed using aequorin as a reporter enzyme. . The aim of this paper is to describe the construction of several eukaryotie promoter-aeq eDNA fusion genes and to demonstrate that the aequorin assay is as reliable and sensitive as the CAT assay and even more advantageous over the previous assays. Another lueiferase (Vargula lueiferase) to serve as a reporter is described in an accompanying paper in this issue (Thompson et al., 19901.
RESULTS AND DISCUSSION
(a) Construction of aaeq eDNA expression plasmids The aaeq expression plasmid, pSV2AQ and its derivatives, pSVOAQ, pSV1AQ, pRSVAQ, pMTAQ, pTKAQ were constructed (Fig. IA). In brief, a D N A fragment containing SV40 small-t intron and polyadenylation signal isolated from pSV2gpt (Mulligan and Berg, 1981) and an aaeq eDNA fragment of 769 bp (Inouye et al., 1985) were ligated to pUC19 to gener~ae pSVOAQ. Then, several eukaryotic promoters were inserted into pSVOAQ. The SV40 early promoter, the LTR of RSV, the mouse MT-I promoter and the HSV-1 tk promoter were obtained from pSV2gpt (Mulligan and Berg, 1981), pSRI (Yamamoto etal., 1980), pMPA-F (Sasaki etal., 1986) and pXl (Enquist et al., 1979), respectively. To obtain an enhancerless expression plasmid (pSV IAQ), both the 55-bp and the 216-bp 5:phI fragments were removed from pSV2AQ. The aaeq eDNA used in this study contained a G stretch , of 33 nt and an out-of-frame AUG codon in the 5'-untranslated region. These sequences may reduce the expression of aaeq cDNA (Cullen, 1987; Kozak, i987). Therefore, pSV2AQA5 and its cnhancerless derivative pSVIAQA5 were constructed, in which the G stretch and the ATG sequence were deleted (Fig. 1B and C).
(b) Linearity and sensitivity of the aequorin assay The relationship between the amount of cell extract and the aequorin activity was investigated by transfecting CV-1 cells with pSV2AQ. As shown in Fig. 2, the aequorin activity was prot: ortional to the amount of extract protein over a 1000-fold range. Extracts from mock transfected cells gave no detectable luminescence. The sensitivity of the assay was limited only by the noise of the photomultiplier, which was probably responsible for the nonlinearity of the curve observed at the lower protein range.
(c) Expression of aaeq cDNA under the control of various promoters To see whether the aequorin assay properly reflects the promoter activities, we transfected CV-I and HeLa cells with the aaeq-expression plasmids shown in Fig. 1 and cat-expression plasmids. At 36h after transfection, ~iequorin and CAT activities in the cell extracts were meastfi"ed. "The results are summarized in Table I. These expresS,ion plasmids appear to be more active in CV-1 than in HeLa.'eells. A promoter-less plasmid pSVOAQ gave almost background value as expected, pSV1 AO, which has the SV40 early promoter, and pSV2AQ, which nas both the SV40 early promoter and the enhancer, gave reasonable levels of expression. An improved expression plasmid, pSV2AQA5, gave higher activity than pSV2 ~Q, indicating that the G-stretch and the out-of-frame AUG affected the efficiency of gene expression as reported (Cu!!en, 1987; Kozak, 1987). Relative activities of various promoters monitored by the aequorin assay were in general agreement with those obtained by the CAT assay, indicating that aequorin may serve as a reliable reporter enzyme for the study of eukaryotic gene expression.
(d) Expression of aaeq in various cell lilies The relative efficiency of the expression of aaeq eDNA was compared in several cell lines transfected with pSV2AQA5 (Table II). Aequorin activity was detected in all the cell lines tested, although the expression in H35-P15 was extremely low. Since H35-PI5 cells transfected with pSV2CAT also showed a very low CAT activity (data riot shown), it is conceivable that successful transfection by the Ca. phosphate method is difficult to achieve with this cell line. The highest activity was observed in COS cell extracts.
Fig. 1. Schematic representation of structure of apoaequorin expression plasmids. (A)Plasmids with various promoters. (B) Structures of improved expression plasmids pSV2AQA5and pSVIAQA5,pSV2AQ has a 33-bp O tail and an out-of-frame AUG codon at the 5' end which are deleted in pSV2AQA5.A SphI-PstI fragment of pSV2AQA5was introduced into pUCI9 to generate an improved enhancerless expresston vector, pSVIAQA5. Details of the procedure for constructingthese plasmids are available on request. (C) The nt sequences of the translation initiation regionsof pSV2AQ and pSV2AQA5.The vertical arrow shows the presumed N terminus of native apoaequorin.
252 TABLE I
I0 2
Relative activities ofaequorin and CAT produced in CV-1 and HeLa cells Plasmid lO m L_ w
m 4~ 0 0
I0
pSV2AQ " pSVIAQ pSVOAQ pRSVAQ pMTAQ pTKAQ pSV2AQd5
0
C 0 O" ee
I0
I0
-I
-2 ' 10 -2
Amount
I
'
I O "1
of
cell
I
'
I
!0 0
!0 1
extract
protein
'
I 10 2
(jxg)
Fig. 2. Relation between the amount of cell extract protein and aequorin activity. Plasmid pSV2AQ DNA was prepared by the modified alkaline lysis method (Maniatis et al., 1982) with polyethyleneglycol precipitation, followed by CsCl-ethidium bromide density gradient equilibrium centrifugation and two cycles of ethanol precipitation. The plasmid DNA preparation was checked by agarose gel electrophoresis to ensure that most plasmids were in the form I configuration. CV-I cells were grown in Dulbecco's Eagle's medium (Nissui Pharmaceutical Co., Tokyo), supplemented with 10% (v/v) heat-treated fetal calf serum (Gibco Laboratories, Detroit, MI) and 80 IUPn/ml. The culture was incubated at 37°C in a humidified atmosphere containing 5% CO2. At 24 h before transfection, cells were plated at a density of 8 x l0 s cells per 100-mm Falcon petri dish, and at 3 h before transfection cells were replenished with fresh medium. Cells were transfected with 20 gg of pSV2AQ DNA per dish using the Ca. phosphate precipitation technique, essentially as described by German (1985). Cells were washed once {4 h later) with PBS( - ). The cells were then placed in 3ml of 157.o glycerol in TBS (0.137M NaCI/5 mM KC1/0.28 mM NaaHPO4/24.8 mM Tris.HCl pH 7.4) per dish and incubated for 2 min at 37°C. It is importaat to remove any Ca" phosphate precipitate, since Ca a + prevents apoaequorin from being converted to active aequorin during the assay. Thus, cells were washed tree times with PBS(- ) and then replenished with fresh medium and incubated at 37°C. A, 36 h after transfection, cells were washed three times with PBS( - ) and suspended in 1 ml of PBS( - ). After centrifugation at 2000 x g for I rain, the pelleted cells were resuspended in 200 #! of extraction buffer A (30 mM Tris. HCi pH 7.6/10 mM EDTA) and lysed by three cycles of freezing m a mixture of dry-ice and methanol and thawing at 37°C. Cell debris were removed by centrifugation (12000 x g, 10 min, 4 ° C), and the resultant supernatants (cell extracts) were used for each assay. The total amount of protein in the extracts was determined by the Coomassie brilliant blue G250 binding assay method (Bradford, 1976), using Bio-Rad reagent (Bio-Rad Laboratories, Richmond, CA). The cell extracts can be stored at -70°C. The aequorin activity was measured as following: Coelenterazine [2-(p-hydroxybenzyl)-6-(p -hydroxyphenyl)-3,7-dihydroimidazo[l,2.a].pyrazin.3.one], a cofactor for aequorin, was chemically synthesized according to the method of lnoue et al. (1975). Apoaequorin in the extracts was converted to active
Aequorin activity ~ Plasmid b CV-I
Hel.a
100 7.4 0.3 271 60 7 181
100 N.D. N.D. 210 68 4 179
CAT activity c CV-1
pSV2CAT pSV 1CAT
100 4.7
pURSVCAT
208
" Relative activity expressed as percentage of the activity from pSV2AQ. Each value was the average of the results of three independent transfaction experiments carried out in parallel. N.D., not determined. HeLa c =lls were grown in Dulbecco's modified Eagle's medium (Nissui Pharmaceutical Co.) supplemented with 10% (v/v) heat-treated fetal calf serum and 80 IUPn~/m!. b CAT-encoding plasmids described by German et al. (1982) were used, excepf for pURSVCAT. Plasmid pURSVCAT was constructed as follows: The HindIII-Bam HI 1632-bp DNA fragment containing the CATcoding sequence and the BstNI-PvulI 524-bp fragment containing LTR of RSV were obtained from pSV2CAT (German et ai., 1982) and pSRI (Yamamoto et al., 1980), respectively. The RSV LTR fragment and the cat fragment were blunted with Klenow enzyme and introduced into the Smal site and the previously blunted Eco Rl site of pUCI9, respectively. c Rel.ative activity expressed as percentage of the activity from pSV2CAT. Each value was the average of triplicate experiments.
This is presumably due to the fact that the plasmid replicates in COS cells (Gluzman, 1981). Since apoaequorin lacks a signal peptide, it was not secreted into the medium (data not shown). The expression of aaeq eDNA in stable transformants was also examined. CV- 1 cells were cotransfected with 1/,g of pSV2 nee (Southern and Berg, 1982) and 19/~g of pSV2AQ by the Ca. phosphate coprecil~it~,tion technique. At 72 h after transfection, the cells were plated at a density
aequorin by incubating the cell extracts on ice with 1 #g ofcoelenterazine (l/~g/#! absolute methanol) in 30mM Tris-HCl pH 7.6/10mM EDTA/2 mM dithiothreitol (or 2~o 2-mercaptoethanol) (final volume of 100/~! in a 1,5-ml Eppendorf tube) (Shimomura and Johnson, 1975; Inouye et al., 1986). The mixture was conventionally incubated overnight to complete the regeneration, but a 5-6 h incubation usually suffices for the regeneration. The mixture was then transferred to a sample tube, which was placed in the holder of a Model TD-4000 Lumiphotometer (Labo-Science, Tokyo) and the initial maximal light intensity was measured after the injection of 600/d of 30 mM CaCl 2 in 30 mM Tris-HCl (pH 7.6). The optimum concentration of CaCl2 for the assay was 10-30 mM. The light intensities were recorded with a Model NP-10!24 strip- chart recorder {Rikadenki, Tokyo), and the activii:y was expressed as relative light units (rlu). One rlu is equivalent to the intensity emitted by 2.5 pg of native aequorin. Each value is the average of three independent experiments.
253 TABLE II
tO I
Aequorin activity in extracts from various cell lines a Cell line
Aequorin activity (rlu)/10 #g of cell-extract protein
m t_
o
I0
CV-I HeLa COS CHO-K1 H35-P! 5 b
3.10 1.92 26.3 13.2 0.024
a COS cells were grown in Dulbecco's modified Eagle's medium (Nissui Pharmaceutical Co.), rat Reuber hepatoma H35-P! 5 cells in Dulbecco's Eagle's medium supplemented with 0.1~ Bacto-peptone (DIFCO, Detroit, MI) and CHO-KI cells in Ham medium (Nissui Pharmaceutical Co.). Each medium was supplemented with 10% (v/v) heat treated fetal calf serum and 80 IUPn/ml. The growth conditions, preparation of cell extracts and the aequorin assay were carried out as described in the legend for Fig. 2. b In the case of H35-PlS, 100 #g of ceil-extract protein was assayed, and the value was calculated as that of 10 #g of cell-extract protein.
10 2
(o) 0
tO I
m 4~
qa
o u
i0 "1
10 0
tO"2
I01
-2
'
i
I0 -I
'
i
I0
O '
: I
tO
10
2
,,,,,:~
18"1 tO t
(b) i L_ v gll 4.1 .m g~ u 411 u
0
i00
tO
iO "1
io-i
'-, ::11
o
of 1 x 106 cells/100-mm dish and maintained for 26 days in a medium containing antibiotic G418 (400 #g/ml). Cell extracts prepared from several independent dishes showed about 3-18~ of the aequorin activity obtained in the transient expression experiments. Thus, aequorin can also be used as a reporter enzyme to monitor gene expression in stable transformants.
(e) Comparison of the aequorin assay with other reporter enzyme systems To compare the sensitivity and linearity of the aequorin assay with those of the CAT and firefly luciferas~, assays, CV-1 cells were cotransfected with an equal amount (10 ~g each) of pSV2AQA5 and pSV2CAT encoding CAT as a reporter enzyme or pSV2AQ,a3 and pSV2ALA5' encoding luciferase as a reporter enzyme (de Wet et al.~ 1987). These plasmids possessed identical SV40 early promoter and enhancer sequences. The activity of each enzyme in the cell extract was found to reach a maximum level at 36 h ~ffter transfection (data not shown). Fig. 3 (a and b) shows the relationship between the enzyme activities and the amounts of cell extracts. The enzyme activities were proportional to the amount of cell extracts above the range of 0.2 #g protein for the CAT assay, about 0.1-0.2/ag of protein for the aequorin assay and 0.1 #g or less of protein for the lueiferase assay. The extract containing 0.1 #g protein from mock transfected cells showed a CAT activity of 0.3~ conversion. Thus, in the CAT assay, the linearity was affected by the background activity. In contrast, neither aequorin nor luciferase activity was found in mock transfected cell extracts so that both assays maintained linearity down to about 0.1 #g cell-extract protein. Hence, the lower
ID lid o o" Q
~ _J
IO-2
10"2 10 .2 Rmot,.~t
I0"1
10 0
I0 I
o f cola ~,n~m~.c| p r o t e i n
102 (jA 9)
Fig. 3. Comparison o;'aequorin, CAT and firefly luciferase assays. CV-I cells were cotransfected with (a)10pg of pSV2AQA5 and 10pg of pSV2CAT or (b) 10 pg of pSV2AQA5 and 10 #g of pSV2AQAS'. Cell extracts were prepar,'d at 36 h after transfection as described above except that t ~ extraction buffer B (250 mM Tris. HCI pH 7.8) for CAT assay and th,~ extraction buffer C (100 mM K. phosphate pH 7.8/1 mM D T I ) lbr luciferase assay were used instead of the extraction buffer A. CAT assay was carried out essentially according to Gorman et al. (1982). The reactions were carried out for 30 rain at 37°C in 0.25 M Tris-HCl pH 7.8/0.1 pCi of [x4C]chloramphenicol (New England Nuclear)/0.44 mM acetyl coenzyme A (Sigma Chemicals) in a total volume of 180 #1. The data were calculated as percent conversion of chloramphenicoi to its acetylated forms. The luciferase assay was carried out according to de Wet et al. (1987). Luciferase activity was proportional to the observed initial maximal light intensity. 1"7,aequorin activity, 0 , CAT activity, l , luciferase activity.
limit of the aequorin and luciferase assays is dependent on the noise level of the photomultiplier employed as mentioned above. The aequorin assay was as sensitive as the CAT assay (Fig. 3a), but the sensitivity of the luciferase assay was several-fold higher than that of the aequorin assay (Fig. 3b). Time co,,rse studies have shown that the expression offirefly luciferase begins to decline significantly in HeLa cells 10 h after transfection (Maxwell and Maxwell,
254 1988). Aequorin seems more stable than the luciferase so that aequorin may be more useful than firefly luciferase in some cases. (f) Conclusions
The present work demonstrates the utility of a photoprotein, aequorin, as a reporter enzyme in studies of eukaryotic gene expression. The aequorin assay is as sensitive as the CAT assay, and it has some advantages over the CAT assay. The aequorin assay does not require the use of radioactive compounds and most of the steps can be carried out only in one or two test tubes. Endogenous enzyme activities often interfere with the assays using CAT, alkaline phosphatase and fl-galactosidase as reporter enzymes, but no endogenous aequorin activity has been detected in mammalian cells. Thus, in practice, the aequorin assay is sensitive, simple and nonhazardous. One of the problems of the aequorin assay is the requirement for coelenterazine, which serves as the substrate. The compound is not commercially available, but it can be chemically synthesized I,Inoue et al., 1975) and should become eventually available. The quantum yield of the firefly luc~.ferase reaction is 0.88 (Seliger and McElroy, 1960) with respect to luciferin, b u t that of aequorin is 0.15 with respect to coelenterazine. This may explain, in part, the difference in the sensitivities of the two assays. Aequorin expression vectors constructed in the present work can be used for various purposes. The plasmid pSVOAQ, lacking a promoter sequence, may be helpful in identifying promoter sequences, whereas pSVIAQ (and pSV1AQA5) containing the SV40 early promoter region may aid in identifying an enhancer sequence. On the other hand, pRSVAQ and pSV2AQ (and pSV2AQA5) may be employed as an internal marker for estimating the transruction efficiency. Finally, the aeq gene appears to have the potential value as a reporter gene in stably transformed cell lines.
ACKNOWLEDGEMENTS We thank Drs. Masahira Hattori and Ken-ichi Umene for plasmids pSV2ALA5' and p X l , respectively. We also thank Prof. Yukio Ikehara and Dr. Yoshio Misumi for advice in culturing H35-PI5 cells, and Misses Michiko Mine and Akiko Kato for their excellent assistance in preparation of the manuscript. This work was supported in part by a grant-in-aid (62105001) from the Ministry of Education, Culture and Science of Japan (to Y.S.) and by research grants (DMB 85-17578 and D M B 88-44459)from the National Science Foundation (to F.I.T.).
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of medusae, with special reference to the photoprotein aequorin. Methods Enzymol. 57 (1978) 271-291. Kozak, M.: An analysis of 5'-noncoding sequences from 699 vertebrate mRNAs. Nucleic Acids Res. 15 (1987) 8125-8148. Maniatis, T., Fritsch, E.F. and Sambrook, J.: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982. Maxwell,I.H. and Maxwell,F.: Electroporation of mammaliancells with a fireflyluciferaseexpression plasmid: Kinetics of transient expression differ markedly among cell types. DNA 7 (1988) 557-562. Mulligan, R.C. and Berg, P.: Selection for animal cells that express the Escherichia coil gene coding for xanthine-guanine phosphoribosyltransferase. Proc. Natl. Acad. Sci. USA 78 (1981) 2072-2076. Sasaki, H., Tone, S., Nakazato, M., Yoshioka, K., Matsuo, H., Kato, Y. and Sakaki, Y.: Generation of transgenic mice producing a human transthyretin variant: A possible mouse model for familialamyloidotic polyneuropathy. Biochem. Biophys. Res. Lommun. 139 (1986) 794-799.
255 Selden, R.F., Howie, K.B., Rowe, M.E., Goodman, H.M. and Moore, D.M.: Human growth hormone as a reporter gene in regulation studies employingtransient gene expression. Mol. Cell. Biol. 6 (1986) 3173-3179. Seliger, H.H. and McElroy, W.D.: Spectral emission and quantum yield of firefly bioluminescence. Arch. Biochem. Biophys. 88 (1960) 136-141. Shimomura, O., and Johnson, F.H.: Calcium binding, quantum yield, and emitting molecule in aequorin bioluminescence. Nature 227 (1970) 1356-1357. Shimomura, O. and Johnson, F.H.: Regeneration of the photoprotein aequorin. Nature 256 (1975) 236-238. Southern, P.J. and Berg, P.: Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J. Mol. Appl. Genet. 1 (1982) 327-341.
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249
GENE 03803
Photoprotein aequorin: use as a reporter enzyme in studying gene expression in mammalian cells (Bioluminescence; calcium ion; CAT assay; eukza,yotic vectors; mammalian geae promoters; recombinant DNA; transfection)
Hiroshi Tanahashi', Takashi Ito b, Satoshi Inouye c, Frederick I. Tsuji d,e and Yoshiyuki Sakaki a ° Research Laboratory for Genetic Information, Kyushu University, Fukuoka 812 (Japan); ~ Institute of Tropical Medicine, Nagasata" University, Nagasaki 852 (Japan) Te1.(0958)47-2111; c Chisso Corporation, Kamaridani-2, Yokohama 236 (Japan) Te1. (045) 701-2443 ; d Osaka Bioscience Institute, Suita, Osaka 565 (Japan) Tel. (06)872-481.~; and e Marine Biology Research Division, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA 92093 (U.S.A.) Tel. (619)534-4245 Received by A. Nakazawa: 9 April 1990 Revised: 23 May 1990
Accepted: 25 May 1990
SUMMARY
Aequorin is a luminescent protein present in the jellyfish Aequoria victoria which emits light (at 460 nm) in the presence of Ca 2 +. We report here that aequorin can be used as a reporter enzyme to monitor gene expression in eukaryotic cells. A cDNA encoding apoaequorin was fused to several eukaryotic !,romoters, including those of SV40, RSV and the HSV-I tk gene, and introduced into several cell lines such as CV-I, COS and HeLa. At appropriate times after transfection, the aequorin activities in cell extracts were measured by monitoring the intensity of fight emitted at 460 nm when triggered by Ca 2 + by the use of a photomultiplier photometer. The aequorin assay was shown to be as sensitive as the conventional CAT assay, and the relative activities of various promoters estimated by the aequorin assay were in general agreement with those obtained by the CAT assay. The aequorin assay c~m be done within 6-7 h from the preparation of extract to the measurement of activity without using radioactive compounds.
INTRODUCTION
Currently, the regulation of eukaryotic gene expression is one of the most important and attractive subjects of study in molecular biology. For the analysis of the regulatory Correspondenceto: Dr. Y. Sakaki, Research Laboratory for Genetic Information, Kyushu Univecsity 3-1-1 Maidashi, Higashi-ku, Fukuoka 812 (Japan) Tel. (092)641-1151 ext. 3461; Fax (092)632-2375. Abbreviations: aaeq, gene (DNA) encoding apoaequorin; aeq, gene (DNA) encoding aequorin; bp, base pair(s); Cm, chloramphenicol; CAT, Cm acetyltransferase; HSV-I, Herpes simplex virus type I; IU, international unit(s); LTR, long terminal repeat; MT-I, gene encoding methailothionein I; nt, nucleotide(s); PBS(-), Ca 2+- and Mg2+-free phosphate-buffered saline (8 g NaCI/0.2 g KCI/I.15 g Na2HPO4/0.02 g KH2PO4 per liter of distilled water); Pn, penicillin G potassium salt; rlu, relative light unit(s); RSV, Rous sarcoma virus; SV40, simian virus 40; tk, gene encoding thymidine kinase. 0378-1119/90/$03.50 © 1990 Elsevier SciencePublishers B.V. (BiomedicalDb,ision)
mechanism, an appropriate reporter gene is often fused to possible regulatory sequences, and the resultant fusion gene is introduced into eukaryotic cells to monitor the effects of the sequences on gene expression. Several reporter genes have been used for this purpose, s~lch a-~those for chloramphenicol acetyltransferase (CAT) (German et al., 1982), //-galactosidase (An et al., 1982), human growth h~rmone (Selden et al., 1986), firefly luciferase (de Wet et aL, 1987) and alkaline phosphatase (Berger et al., 1988; Henthorn et al., 1988; Yoon et al., 1988). We have previously isolated a full-length aeq cDNA, encoding aequorin, a luminescent protein which emits light with a peak at 460 nm in the presence of Ca 2 + (Inouye et al., 1985; Tsuji et al., 1986): aequorin 3 caz+ ~hv + CO2 + apoaequorin+ coelenteramide ~ _ [ coelenterazine+ EDTA +'~ I O2 + 2-mercaptoethanol _!
250
(k) PMI
Pstl
~
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.Kpnl
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n,,,u,,,
I "
~
pSV2AQ
| | Kpnl
)
Barn H HI
Barn Hi I "~mmm"~ BamHI " His dill
Pstl
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Barn HI Hlnd;ll Sphl Pstl Sail EcoR Kpnl K/
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pSV?gpt-dedved SV40 smsll-t Inlron end polysdenylstlon signal ~
LTR of RSV
,.-od:ng sequence olaaeacONA
~
MTJ promoter
non-ceding sequence of aaeacDNA
~
SV40early promoter
HSV-1 t k gene promoter
(C) Qpoaequorln N-terminus
psv2Ao
V';,, ] ~let Thr Set LUs Gin Tyr Se LUs ....... 60bp ........ AACAA6CAA~C|ATG Al:h AGe AAA CAATACTC, ~T: AAG .........
~(G)/~T.~t;AATT ,
.
.......I.''D''''" .oD''" O.,,,,,,.,.I.D''''
251 Aequorin seems to have some advantages as a reporter enzyme. For example, the quantum yield of the reaction is reasonably high (0.15) (Shimomura and Johnson, 1970; Johnson and Shimomura, 1978), and the assay of aequorin can be done within several h from the preparation of cell extracts to the end of assay withota using radioactive compounds. The activity can be easily and quantitatively measured by using a conventional photomultiplier photometer. Thus, a unique system can be developed using aequorin as a reporter enzyme. . The aim of this paper is to describe the construction of several eukaryotie promoter-aeq eDNA fusion genes and to demonstrate that the aequorin assay is as reliable and sensitive as the CAT assay and even more advantageous over the previous assays. Another lueiferase (Vargula lueiferase) to serve as a reporter is described in an accompanying paper in this issue (Thompson et al., 19901.
RESULTS AND DISCUSSION
(a) Construction of aaeq eDNA expression plasmids The aaeq expression plasmid, pSV2AQ and its derivatives, pSVOAQ, pSV1AQ, pRSVAQ, pMTAQ, pTKAQ were constructed (Fig. IA). In brief, a D N A fragment containing SV40 small-t intron and polyadenylation signal isolated from pSV2gpt (Mulligan and Berg, 1981) and an aaeq eDNA fragment of 769 bp (Inouye et al., 1985) were ligated to pUC19 to gener~ae pSVOAQ. Then, several eukaryotic promoters were inserted into pSVOAQ. The SV40 early promoter, the LTR of RSV, the mouse MT-I promoter and the HSV-1 tk promoter were obtained from pSV2gpt (Mulligan and Berg, 1981), pSRI (Yamamoto etal., 1980), pMPA-F (Sasaki etal., 1986) and pXl (Enquist et al., 1979), respectively. To obtain an enhancerless expression plasmid (pSV IAQ), both the 55-bp and the 216-bp 5:phI fragments were removed from pSV2AQ. The aaeq eDNA used in this study contained a G stretch , of 33 nt and an out-of-frame AUG codon in the 5'-untranslated region. These sequences may reduce the expression of aaeq cDNA (Cullen, 1987; Kozak, i987). Therefore, pSV2AQA5 and its cnhancerless derivative pSVIAQA5 were constructed, in which the G stretch and the ATG sequence were deleted (Fig. 1B and C).
(b) Linearity and sensitivity of the aequorin assay The relationship between the amount of cell extract and the aequorin activity was investigated by transfecting CV-1 cells with pSV2AQ. As shown in Fig. 2, the aequorin activity was prot: ortional to the amount of extract protein over a 1000-fold range. Extracts from mock transfected cells gave no detectable luminescence. The sensitivity of the assay was limited only by the noise of the photomultiplier, which was probably responsible for the nonlinearity of the curve observed at the lower protein range.
(c) Expression of aaeq cDNA under the control of various promoters To see whether the aequorin assay properly reflects the promoter activities, we transfected CV-I and HeLa cells with the aaeq-expression plasmids shown in Fig. 1 and cat-expression plasmids. At 36h after transfection, ~iequorin and CAT activities in the cell extracts were meastfi"ed. "The results are summarized in Table I. These expresS,ion plasmids appear to be more active in CV-1 than in HeLa.'eells. A promoter-less plasmid pSVOAQ gave almost background value as expected, pSV1 AO, which has the SV40 early promoter, and pSV2AQ, which nas both the SV40 early promoter and the enhancer, gave reasonable levels of expression. An improved expression plasmid, pSV2AQA5, gave higher activity than pSV2 ~Q, indicating that the G-stretch and the out-of-frame AUG affected the efficiency of gene expression as reported (Cu!!en, 1987; Kozak, 1987). Relative activities of various promoters monitored by the aequorin assay were in general agreement with those obtained by the CAT assay, indicating that aequorin may serve as a reliable reporter enzyme for the study of eukaryotic gene expression.
(d) Expression of aaeq in various cell lilies The relative efficiency of the expression of aaeq eDNA was compared in several cell lines transfected with pSV2AQA5 (Table II). Aequorin activity was detected in all the cell lines tested, although the expression in H35-P15 was extremely low. Since H35-PI5 cells transfected with pSV2CAT also showed a very low CAT activity (data riot shown), it is conceivable that successful transfection by the Ca. phosphate method is difficult to achieve with this cell line. The highest activity was observed in COS cell extracts.
Fig. 1. Schematic representation of structure of apoaequorin expression plasmids. (A)Plasmids with various promoters. (B) Structures of improved expression plasmids pSV2AQA5and pSVIAQA5,pSV2AQ has a 33-bp O tail and an out-of-frame AUG codon at the 5' end which are deleted in pSV2AQA5.A SphI-PstI fragment of pSV2AQA5was introduced into pUCI9 to generate an improved enhancerless expresston vector, pSVIAQA5. Details of the procedure for constructingthese plasmids are available on request. (C) The nt sequences of the translation initiation regionsof pSV2AQ and pSV2AQA5.The vertical arrow shows the presumed N terminus of native apoaequorin.
252 TABLE I
I0 2
Relative activities ofaequorin and CAT produced in CV-1 and HeLa cells Plasmid lO m L_ w
m 4~ 0 0
I0
pSV2AQ " pSVIAQ pSVOAQ pRSVAQ pMTAQ pTKAQ pSV2AQd5
0
C 0 O" ee
I0
I0
-I
-2 ' 10 -2
Amount
I
'
I O "1
of
cell
I
'
I
!0 0
!0 1
extract
protein
'
I 10 2
(jxg)
Fig. 2. Relation between the amount of cell extract protein and aequorin activity. Plasmid pSV2AQ DNA was prepared by the modified alkaline lysis method (Maniatis et al., 1982) with polyethyleneglycol precipitation, followed by CsCl-ethidium bromide density gradient equilibrium centrifugation and two cycles of ethanol precipitation. The plasmid DNA preparation was checked by agarose gel electrophoresis to ensure that most plasmids were in the form I configuration. CV-I cells were grown in Dulbecco's Eagle's medium (Nissui Pharmaceutical Co., Tokyo), supplemented with 10% (v/v) heat-treated fetal calf serum (Gibco Laboratories, Detroit, MI) and 80 IUPn/ml. The culture was incubated at 37°C in a humidified atmosphere containing 5% CO2. At 24 h before transfection, cells were plated at a density of 8 x l0 s cells per 100-mm Falcon petri dish, and at 3 h before transfection cells were replenished with fresh medium. Cells were transfected with 20 gg of pSV2AQ DNA per dish using the Ca. phosphate precipitation technique, essentially as described by German (1985). Cells were washed once {4 h later) with PBS( - ). The cells were then placed in 3ml of 157.o glycerol in TBS (0.137M NaCI/5 mM KC1/0.28 mM NaaHPO4/24.8 mM Tris.HCl pH 7.4) per dish and incubated for 2 min at 37°C. It is importaat to remove any Ca" phosphate precipitate, since Ca a + prevents apoaequorin from being converted to active aequorin during the assay. Thus, cells were washed tree times with PBS(- ) and then replenished with fresh medium and incubated at 37°C. A, 36 h after transfection, cells were washed three times with PBS( - ) and suspended in 1 ml of PBS( - ). After centrifugation at 2000 x g for I rain, the pelleted cells were resuspended in 200 #! of extraction buffer A (30 mM Tris. HCi pH 7.6/10 mM EDTA) and lysed by three cycles of freezing m a mixture of dry-ice and methanol and thawing at 37°C. Cell debris were removed by centrifugation (12000 x g, 10 min, 4 ° C), and the resultant supernatants (cell extracts) were used for each assay. The total amount of protein in the extracts was determined by the Coomassie brilliant blue G250 binding assay method (Bradford, 1976), using Bio-Rad reagent (Bio-Rad Laboratories, Richmond, CA). The cell extracts can be stored at -70°C. The aequorin activity was measured as following: Coelenterazine [2-(p-hydroxybenzyl)-6-(p -hydroxyphenyl)-3,7-dihydroimidazo[l,2.a].pyrazin.3.one], a cofactor for aequorin, was chemically synthesized according to the method of lnoue et al. (1975). Apoaequorin in the extracts was converted to active
Aequorin activity ~ Plasmid b CV-I
Hel.a
100 7.4 0.3 271 60 7 181
100 N.D. N.D. 210 68 4 179
CAT activity c CV-1
pSV2CAT pSV 1CAT
100 4.7
pURSVCAT
208
" Relative activity expressed as percentage of the activity from pSV2AQ. Each value was the average of the results of three independent transfaction experiments carried out in parallel. N.D., not determined. HeLa c =lls were grown in Dulbecco's modified Eagle's medium (Nissui Pharmaceutical Co.) supplemented with 10% (v/v) heat-treated fetal calf serum and 80 IUPn~/m!. b CAT-encoding plasmids described by German et al. (1982) were used, excepf for pURSVCAT. Plasmid pURSVCAT was constructed as follows: The HindIII-Bam HI 1632-bp DNA fragment containing the CATcoding sequence and the BstNI-PvulI 524-bp fragment containing LTR of RSV were obtained from pSV2CAT (German et ai., 1982) and pSRI (Yamamoto et al., 1980), respectively. The RSV LTR fragment and the cat fragment were blunted with Klenow enzyme and introduced into the Smal site and the previously blunted Eco Rl site of pUCI9, respectively. c Rel.ative activity expressed as percentage of the activity from pSV2CAT. Each value was the average of triplicate experiments.
This is presumably due to the fact that the plasmid replicates in COS cells (Gluzman, 1981). Since apoaequorin lacks a signal peptide, it was not secreted into the medium (data not shown). The expression of aaeq eDNA in stable transformants was also examined. CV- 1 cells were cotransfected with 1/,g of pSV2 nee (Southern and Berg, 1982) and 19/~g of pSV2AQ by the Ca. phosphate coprecil~it~,tion technique. At 72 h after transfection, the cells were plated at a density
aequorin by incubating the cell extracts on ice with 1 #g ofcoelenterazine (l/~g/#! absolute methanol) in 30mM Tris-HCl pH 7.6/10mM EDTA/2 mM dithiothreitol (or 2~o 2-mercaptoethanol) (final volume of 100/~! in a 1,5-ml Eppendorf tube) (Shimomura and Johnson, 1975; Inouye et al., 1986). The mixture was conventionally incubated overnight to complete the regeneration, but a 5-6 h incubation usually suffices for the regeneration. The mixture was then transferred to a sample tube, which was placed in the holder of a Model TD-4000 Lumiphotometer (Labo-Science, Tokyo) and the initial maximal light intensity was measured after the injection of 600/d of 30 mM CaCl 2 in 30 mM Tris-HCl (pH 7.6). The optimum concentration of CaCl2 for the assay was 10-30 mM. The light intensities were recorded with a Model NP-10!24 strip- chart recorder {Rikadenki, Tokyo), and the activii:y was expressed as relative light units (rlu). One rlu is equivalent to the intensity emitted by 2.5 pg of native aequorin. Each value is the average of three independent experiments.
253 TABLE II
tO I
Aequorin activity in extracts from various cell lines a Cell line
Aequorin activity (rlu)/10 #g of cell-extract protein
m t_
o
I0
CV-I HeLa COS CHO-K1 H35-P! 5 b
3.10 1.92 26.3 13.2 0.024
a COS cells were grown in Dulbecco's modified Eagle's medium (Nissui Pharmaceutical Co.), rat Reuber hepatoma H35-P! 5 cells in Dulbecco's Eagle's medium supplemented with 0.1~ Bacto-peptone (DIFCO, Detroit, MI) and CHO-KI cells in Ham medium (Nissui Pharmaceutical Co.). Each medium was supplemented with 10% (v/v) heat treated fetal calf serum and 80 IUPn/ml. The growth conditions, preparation of cell extracts and the aequorin assay were carried out as described in the legend for Fig. 2. b In the case of H35-PlS, 100 #g of ceil-extract protein was assayed, and the value was calculated as that of 10 #g of cell-extract protein.
10 2
(o) 0
tO I
m 4~
qa
o u
i0 "1
10 0
tO"2
I01
-2
'
i
I0 -I
'
i
I0
O '
: I
tO
10
2
,,,,,:~
18"1 tO t
(b) i L_ v gll 4.1 .m g~ u 411 u
0
i00
tO
iO "1
io-i
'-, ::11
o
of 1 x 106 cells/100-mm dish and maintained for 26 days in a medium containing antibiotic G418 (400 #g/ml). Cell extracts prepared from several independent dishes showed about 3-18~ of the aequorin activity obtained in the transient expression experiments. Thus, aequorin can also be used as a reporter enzyme to monitor gene expression in stable transformants.
(e) Comparison of the aequorin assay with other reporter enzyme systems To compare the sensitivity and linearity of the aequorin assay with those of the CAT and firefly luciferas~, assays, CV-1 cells were cotransfected with an equal amount (10 ~g each) of pSV2AQA5 and pSV2CAT encoding CAT as a reporter enzyme or pSV2AQ,a3 and pSV2ALA5' encoding luciferase as a reporter enzyme (de Wet et al.~ 1987). These plasmids possessed identical SV40 early promoter and enhancer sequences. The activity of each enzyme in the cell extract was found to reach a maximum level at 36 h ~ffter transfection (data not shown). Fig. 3 (a and b) shows the relationship between the enzyme activities and the amounts of cell extracts. The enzyme activities were proportional to the amount of cell extracts above the range of 0.2 #g protein for the CAT assay, about 0.1-0.2/ag of protein for the aequorin assay and 0.1 #g or less of protein for the lueiferase assay. The extract containing 0.1 #g protein from mock transfected cells showed a CAT activity of 0.3~ conversion. Thus, in the CAT assay, the linearity was affected by the background activity. In contrast, neither aequorin nor luciferase activity was found in mock transfected cell extracts so that both assays maintained linearity down to about 0.1 #g cell-extract protein. Hence, the lower
ID lid o o" Q
~ _J
IO-2
10"2 10 .2 Rmot,.~t
I0"1
10 0
I0 I
o f cola ~,n~m~.c| p r o t e i n
102 (jA 9)
Fig. 3. Comparison o;'aequorin, CAT and firefly luciferase assays. CV-I cells were cotransfected with (a)10pg of pSV2AQA5 and 10pg of pSV2CAT or (b) 10 pg of pSV2AQA5 and 10 #g of pSV2AQAS'. Cell extracts were prepar,'d at 36 h after transfection as described above except that t ~ extraction buffer B (250 mM Tris. HCI pH 7.8) for CAT assay and th,~ extraction buffer C (100 mM K. phosphate pH 7.8/1 mM D T I ) lbr luciferase assay were used instead of the extraction buffer A. CAT assay was carried out essentially according to Gorman et al. (1982). The reactions were carried out for 30 rain at 37°C in 0.25 M Tris-HCl pH 7.8/0.1 pCi of [x4C]chloramphenicol (New England Nuclear)/0.44 mM acetyl coenzyme A (Sigma Chemicals) in a total volume of 180 #1. The data were calculated as percent conversion of chloramphenicoi to its acetylated forms. The luciferase assay was carried out according to de Wet et al. (1987). Luciferase activity was proportional to the observed initial maximal light intensity. 1"7,aequorin activity, 0 , CAT activity, l , luciferase activity.
limit of the aequorin and luciferase assays is dependent on the noise level of the photomultiplier employed as mentioned above. The aequorin assay was as sensitive as the CAT assay (Fig. 3a), but the sensitivity of the luciferase assay was several-fold higher than that of the aequorin assay (Fig. 3b). Time co,,rse studies have shown that the expression offirefly luciferase begins to decline significantly in HeLa cells 10 h after transfection (Maxwell and Maxwell,
254 1988). Aequorin seems more stable than the luciferase so that aequorin may be more useful than firefly luciferase in some cases. (f) Conclusions
The present work demonstrates the utility of a photoprotein, aequorin, as a reporter enzyme in studies of eukaryotic gene expression. The aequorin assay is as sensitive as the CAT assay, and it has some advantages over the CAT assay. The aequorin assay does not require the use of radioactive compounds and most of the steps can be carried out only in one or two test tubes. Endogenous enzyme activities often interfere with the assays using CAT, alkaline phosphatase and fl-galactosidase as reporter enzymes, but no endogenous aequorin activity has been detected in mammalian cells. Thus, in practice, the aequorin assay is sensitive, simple and nonhazardous. One of the problems of the aequorin assay is the requirement for coelenterazine, which serves as the substrate. The compound is not commercially available, but it can be chemically synthesized I,Inoue et al., 1975) and should become eventually available. The quantum yield of the firefly luc~.ferase reaction is 0.88 (Seliger and McElroy, 1960) with respect to luciferin, b u t that of aequorin is 0.15 with respect to coelenterazine. This may explain, in part, the difference in the sensitivities of the two assays. Aequorin expression vectors constructed in the present work can be used for various purposes. The plasmid pSVOAQ, lacking a promoter sequence, may be helpful in identifying promoter sequences, whereas pSVIAQ (and pSV1AQA5) containing the SV40 early promoter region may aid in identifying an enhancer sequence. On the other hand, pRSVAQ and pSV2AQ (and pSV2AQA5) may be employed as an internal marker for estimating the transruction efficiency. Finally, the aeq gene appears to have the potential value as a reporter gene in stably transformed cell lines.
ACKNOWLEDGEMENTS We thank Drs. Masahira Hattori and Ken-ichi Umene for plasmids pSV2ALA5' and p X l , respectively. We also thank Prof. Yukio Ikehara and Dr. Yoshio Misumi for advice in culturing H35-PI5 cells, and Misses Michiko Mine and Akiko Kato for their excellent assistance in preparation of the manuscript. This work was supported in part by a grant-in-aid (62105001) from the Ministry of Education, Culture and Science of Japan (to Y.S.) and by research grants (DMB 85-17578 and D M B 88-44459)from the National Science Foundation (to F.I.T.).
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