Biochemical SocietyTransactions (1 992)20 143s Expression of recombinant firefly luciferase in prokaryotic and eukaryotic cells. GRACIELA B. SALA-NEWBY CAMPBELL
and
ANTHONY K.
Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, U.K. Firefly (Photinus pyralis) luciferase has been used to measure ATP and other metabolites in cell extracts. Its use to measure intracellular ATP may be limited because the enzyme is saturated at ATP concentrations found in live cells and it carries a perioxisomal targetting signal at the C-terminus [1,2]. We have engineered it to measure protein kinase A [3]. We also inserted a mitochondrial presequence, removed the peroxisomal signal and showed that the luciferase then localized to the cytosol in COS-7 cells. Two E. coli expression systems were investigated in order to produce protein for microinjection. The polymerase chain reaction was used to remove 9 base pairs at the 3' end of the cDNA coding for SKL, the peroxisomal signal, to add the coding sequence for an artificial mitochondria presequence [4] and to add the T7 RNA polymerase promoter and restriction sites for cloning as previously described 131. The synthetic DNA was transcribed and translated to generate light emitting protein [3]. The catalytic activity of the recombinant proteins were indistinguishable from the wild type. Luciferase with the mitochondrial presequence and wild type synthesized in rabbit reticulocyte were incubated with energized mitochondria C41. The mitochondria bound 10-27 times more of the luciferase with the presequence. A
B l 7 RNA polymerase expression
Expressionvector: pNHl8a ng lwfensslml cullwe 1
I
ng ludferasrnl CUlhlrO
I
1.41
12
80
The cDNA coding for luciferase (minus SKL) was cloned into the plasmid pSV7d [5l. Cos cells transfected with 7 pg of CsCl purified plasmid per cell emitted light when incubated in suspension with luciferin (0.51 mM). We detected (24 i 18) x 106 (n=4) chemiluminescent counts per 10 sec per 106 cells. The chemiluminescence detected in live cells decreased by 81 k 7% (1113) when the cells were heated with oligomycin (2 pg/ml) plus 2-deoxyglucose ( 2 mM) suggesting that the chemiluminescence is proportional to the intracellular ATP concentration. Transfected cells stained with rabbit antiluciferase antibodies and FITC labelled second antibody showed difuse cytoplasmic immunofluorescence in 7% of them. We tested two E. coli expression systems. The pNH 18a plasmid has invertible tandem promoters (ptac-plac). A short heat pulse (10 min at 42OC) inverts the promoter and isopropyl p-D-thiogalactoside (IPTG) derepresses it [ 6 ] . The pSV7d construct carries a T7 promoter upstream of the firefly cDNA and when transfected into E. coli BL21 (DE3) that expresses T7 RNA polymerase under the control of the lac promoter [7] can be induced to produce luciferase. For pNHlBa, 4 clones were studied with 3,6 and 7 in the correct orientation and 2, not (Fig. 1). IPTG increases the luciferase content even when the promoter is in the wrong orientation. Heat shock and IPTG together resulted in a mean increase of 28 times in luciferase production from 3, 6 and 7 (75 ng/ml culture) and 5 times from 2. For T7 RNA polymerase driven expression (Fig. l), IPTG increased the concentration of luciferase 25 times from clone A (1.3 ng/ml/culture) and not at all from clone B. We would like to thank the Medical Research Council for financial support and Andrew Trimby for technical assistance. 1. Campbell, A.K. (1988) Chemiluminescence: principles and applications in biology and medicine p 1-608. Chichester, Weinheim, Horwood, VCH.
1
80
2. Gould, S. J., Keller, G.A., Hosken, N., Wilkinson, J. & Subramani, S. (1989) J. Cell. Biol. 108, 1657-1664.
0.8 4a
0.6
20
0.4
0
02
done number 4PTG.3OC
a 4PTG.30C
0
3. Sala-Newby, G.B. & Campbell, A.K. Biochem. J. 279, 727-732. PSVV"
done
a UC.+IPTG 0
rlPTG.UC
-IPTG
a+IPTG
EiQ.-l A : E. coli Dl2lOHP carrying pNH18a clones were incubated at 30OC in LB broth, 50 pg/ml ampicillin to A650-0.2 k IPTG 1 mM. Heat treatment (10 min at 42OC) and IPTG as indicated followed by 2 hrs at 30OC. B : E. coli EL21 (DE3) transfected with pSV7d constructs were incubated at 37OC in LB broth, 50 pg/ml ampicillin to A650=0.6. IPTG (1 mM) was added as indicated and incubation continued for 3 hrs. Luciferase was extracted by lysozyme (2 mg/ml) digestion in 50 mM TRIS, 2 mM EDTA, 0.2 mg/ml protamine sulfate and assayed as described [3].
(1991)
4 . Allison, D.S.
C Schatz, G. (1986) Proc. Natl. Acad. Sci., USA 83, 9011-9015.
5. Truett, M.A., Blacher, R., a1 (1985) DNA 4, 333-349.
Burke, R.L. et
6. Hasan, N. and Szybalski, W. (1987) Gene 56, 145-151. 7. Studier, F.W., Rosenberg, A.H., Dunn, J.J. & Dubendorff, J.W. (1990) Methods in Enzymology 185, 60-89.
and
ANTHONY K.
Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, U.K. Firefly (Photinus pyralis) luciferase has been used to measure ATP and other metabolites in cell extracts. Its use to measure intracellular ATP may be limited because the enzyme is saturated at ATP concentrations found in live cells and it carries a perioxisomal targetting signal at the C-terminus [1,2]. We have engineered it to measure protein kinase A [3]. We also inserted a mitochondrial presequence, removed the peroxisomal signal and showed that the luciferase then localized to the cytosol in COS-7 cells. Two E. coli expression systems were investigated in order to produce protein for microinjection. The polymerase chain reaction was used to remove 9 base pairs at the 3' end of the cDNA coding for SKL, the peroxisomal signal, to add the coding sequence for an artificial mitochondria presequence [4] and to add the T7 RNA polymerase promoter and restriction sites for cloning as previously described 131. The synthetic DNA was transcribed and translated to generate light emitting protein [3]. The catalytic activity of the recombinant proteins were indistinguishable from the wild type. Luciferase with the mitochondrial presequence and wild type synthesized in rabbit reticulocyte were incubated with energized mitochondria C41. The mitochondria bound 10-27 times more of the luciferase with the presequence. A
B l 7 RNA polymerase expression
Expressionvector: pNHl8a ng lwfensslml cullwe 1
I
ng ludferasrnl CUlhlrO
I
1.41
12
80
The cDNA coding for luciferase (minus SKL) was cloned into the plasmid pSV7d [5l. Cos cells transfected with 7 pg of CsCl purified plasmid per cell emitted light when incubated in suspension with luciferin (0.51 mM). We detected (24 i 18) x 106 (n=4) chemiluminescent counts per 10 sec per 106 cells. The chemiluminescence detected in live cells decreased by 81 k 7% (1113) when the cells were heated with oligomycin (2 pg/ml) plus 2-deoxyglucose ( 2 mM) suggesting that the chemiluminescence is proportional to the intracellular ATP concentration. Transfected cells stained with rabbit antiluciferase antibodies and FITC labelled second antibody showed difuse cytoplasmic immunofluorescence in 7% of them. We tested two E. coli expression systems. The pNH 18a plasmid has invertible tandem promoters (ptac-plac). A short heat pulse (10 min at 42OC) inverts the promoter and isopropyl p-D-thiogalactoside (IPTG) derepresses it [ 6 ] . The pSV7d construct carries a T7 promoter upstream of the firefly cDNA and when transfected into E. coli BL21 (DE3) that expresses T7 RNA polymerase under the control of the lac promoter [7] can be induced to produce luciferase. For pNHlBa, 4 clones were studied with 3,6 and 7 in the correct orientation and 2, not (Fig. 1). IPTG increases the luciferase content even when the promoter is in the wrong orientation. Heat shock and IPTG together resulted in a mean increase of 28 times in luciferase production from 3, 6 and 7 (75 ng/ml culture) and 5 times from 2. For T7 RNA polymerase driven expression (Fig. l), IPTG increased the concentration of luciferase 25 times from clone A (1.3 ng/ml/culture) and not at all from clone B. We would like to thank the Medical Research Council for financial support and Andrew Trimby for technical assistance. 1. Campbell, A.K. (1988) Chemiluminescence: principles and applications in biology and medicine p 1-608. Chichester, Weinheim, Horwood, VCH.
1
80
2. Gould, S. J., Keller, G.A., Hosken, N., Wilkinson, J. & Subramani, S. (1989) J. Cell. Biol. 108, 1657-1664.
0.8 4a
0.6
20
0.4
0
02
done number 4PTG.3OC
a 4PTG.30C
0
3. Sala-Newby, G.B. & Campbell, A.K. Biochem. J. 279, 727-732. PSVV"
done
a UC.+IPTG 0
rlPTG.UC
-IPTG
a+IPTG
EiQ.-l A : E. coli Dl2lOHP carrying pNH18a clones were incubated at 30OC in LB broth, 50 pg/ml ampicillin to A650-0.2 k IPTG 1 mM. Heat treatment (10 min at 42OC) and IPTG as indicated followed by 2 hrs at 30OC. B : E. coli EL21 (DE3) transfected with pSV7d constructs were incubated at 37OC in LB broth, 50 pg/ml ampicillin to A650=0.6. IPTG (1 mM) was added as indicated and incubation continued for 3 hrs. Luciferase was extracted by lysozyme (2 mg/ml) digestion in 50 mM TRIS, 2 mM EDTA, 0.2 mg/ml protamine sulfate and assayed as described [3].
(1991)
4 . Allison, D.S.
C Schatz, G. (1986) Proc. Natl. Acad. Sci., USA 83, 9011-9015.
5. Truett, M.A., Blacher, R., a1 (1985) DNA 4, 333-349.
Burke, R.L. et
6. Hasan, N. and Szybalski, W. (1987) Gene 56, 145-151. 7. Studier, F.W., Rosenberg, A.H., Dunn, J.J. & Dubendorff, J.W. (1990) Methods in Enzymology 185, 60-89.
