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Cloning of Firefly Luciferase Cdna and the Expression of Active Proc. Natl. Acad. Sci. USA Vol. 82, pp. 7870-7873, December 1985 Biochemistry Cloning of firefly luciferase cDNA and the expression of active luciferase in Escherichia coli (bioluminescence/Photinus pyralis/antibody screening/expression vector/recombinant DNA) JEFFREY R. DE WET*, KEITH V. WOODt, DONALD R. HELINSKI*, AND MARLENE DELUCAt Departments of *Biology and tChemistry, University of California, San Diego, La Jolla, CA 92093 Communicated by W. D. McElroy, July 26, 1985 ABSTRACT A cDNA library was constructed from firefly library was screened with anti-P. pyralis luciferase antibody, (Photinuspyralis) lantern poly(A)I RNA, using the Escherichia using a chromogenic detection technique (8), and several coli expression vector Xgtll. The library was screened with cDNA clones were isolated and characterized. These clones anti-P. pyralis luciferase (Photinus luciferin:oxygen 4-oxidore- were found to be homologous to the mRNA that encodes ductase, EC 1.13.12.7) antibody, and several cDNA clones luciferase. The largest luciferase cDNA clone that was expressing luciferase antigens were isolated. One clone, ALucl, isolated was able to direct the synthesis of active luciferase contained 1.5 kilobase pairs of cDNA that hybridized to a 1.9- in E. coli. to 2.0-kilobase band on a nitrocellulose blot of electrophoreti- cally fractionated lantern RNA. Hybridization of the cloned MATERIALS AND METHODS cDNA to lantern poly(A)I RNA selected an RNA that directed the in vitro synthesis of a single polypeptide. This polypeptide Enzymes and Strains. Restriction endonucleases and E. coli comigrated with luciferase on NaDodSO4/PAGE and produced DNA polymerase I were purchased from New England bioluminescence upon the addition of luciferin and ATP. A Biolabs. RNase H and bacteriophage X in vitro packaging 1.8-kilobase-pair cDNA was isolated by probing the firefly extracts were obtained from Bethesda Research Laborato- cDNA library with the cDNA from XLucl. This cDNA con- ries, and avian myeloblastosis virus reverse transcriptase tained sufficient coding information to direct the synthesis of was from Boehringer Mannheim. XgtJ1 and E. coli strains active firefly luciferase in E. coli. Y1088 [supE supF metB trpR hsdR hsdM' tonA21 AlacUl69 proC:TnS(pMC9)] and Y1090 [AlacUl69 proA' Alon Luciferases are the enzymes that catalyze the light-producing araD139 strA supF trpC22:TnlO(pMC9)] were obtained from chemical reactions of bioluminescent organisms. Insect R. Young (6, 7). E. coli strain TB1 [ara A(lac-proA,B) strA luciferases require ATP, an organic molecule called luciferin, f80dlacZAM15 hsr- hsm'] was obtained from T. Baldwin and oxygen as substrates (1). The absolute requirement for (Texas A & M University). The plasmid pUC13 has been ATP as a substrate is a characteristic unique to insect described (9). The E. coli expression plasmid pKJB824.17, luciferases, and this property has been used to develop consisting of pBR322 carrying the temperature-sensitive X bioluminescence assays for ATP (1). repressor gene c1857 and the X promoter PR was obtained The only insect luciferase that has been purified and from K. Buckley (10). extensively characterized was isolated from the North Amer- Construction and Screening of the Xgtll cDNA Library. ican firefly, Photinus pyralis (Photinus luciferin:oxygen 4- Live fireflies (P. pyralis) were obtained from W. Biggley oxidoreductase, EC 1.13.12.7) (1, 2). This enzyme catalyzes (Johns Hopkins University). Isolated lanterns were homog- the conversion of chemical energy into light with a very high enized in guanidinium thiocyanate, and total lantern RNA efficiency; the quantum yield (photons emitted per molecule was isolated from the homogenate by sedimentation through of luciferin oxidized) is 0.88 (3). Purified P. pyralis luciferase a CsCl cushion (11). Poly(A)+ RNA was isolated by chro- migrates in NaDodSO4/polyacrylamide gels as a single band matography on oligo(dT)-cellulose (12). The construction of at an apparent molecular weight of62,000 (4). The luciferases the Xgtl1 cDNA library was as described (8), except that from other firefly species migrate at a similar position, and all RNase H and DNA polymerase I were used to synthesize the show extensive crossreactivity with antibody raised against second strand of the cDNA (13). Packaging of 1 lg of Xgt1l P. pyralis luciferase (26). P. pyralis emits yellow-green light DNA ligated to cDNA yielded =101 recombinant phage, and with peak emission occurring at 560 nm (3). Other species of after amplification of the library on Y1088, the recombinants fireflies emit different colors oflight ranging from yellow (582 represented --10% of the total phage population. The ampli- nm) to green (552 nm), the particular color being a charac- fied library was screened with rabbit anti-luciferase antibody teristic of a given species (5). Since all firefly luciferases use (2 Ag/ml) as described (8). the same substrates (5), these observed differences indicate Hybridizations. All hybridizations were performed with that the color of light emission is dependent on enzyme isolated restriction fragments used as probe. Restriction structure. Comparative studies of insect luciferases may fragments were isolated from agarose gels by electrophoresis yield information on how changes in protein structure affect onto DE81 paper (14), and the DNA was labeled with the light-emission properties of these enzymes. [a-32P]dCTP (-800 Ci/mmol, Amersham; 1 Ci = 37 GBq) by In an earlier paper we showed that poly(A)+ RNA isolated nick-translation (15) to a specific activity of :-108 cpm/pug. from the lanterns ofadult P. pyralis fireflies contained mRNA Phage were plated on Y1088 cells and screened by plaque that directed the synthesis of luciferase in vitro (4). We have hybridization (16). Southern blots were prepared essentially used this poly(A)+ RNA to construct a cDNA library in as described (17). RNA samples were electrophoresed in Xgtll, an Escherichia coli expression vector (6, 7). The formaldehyde/agarose gels and blotted onto nitrocellulose (Schleicher & Schuell, BA85) (18). Hybridizations ofprobe to The publication costs of this article were defrayed in part by page charge filters were at 37°C in 55% (vol/vol) formamide/5x SSPE (lx payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviations: bp, base pair(s); kb, kilobase(s). 7870 Downloaded by guest on October 1, 2021 Biochemistry: de Wet et al. Proc. NatL. Acad. Sci. USA 82 (1985) 7871 SSPE is 0.18 M NaC/10 mM sodium phosphate, pH 7.7/1 mM 1 2 EDTA)/heparin (200 pug/ml)/0.1% NaDodSO4 containing 2 x 105 cpm of nick-translated probe per ml (19). Filters were washed in 0.1x SSPE/0.1% NaDodSO4 at 370C and autora- 28S- diographed with Kodak XAR-5 film and Cronex Lightning- plus intensifying screens at -70'C. 23S- Hybridization-Selection and in Vitro Translation. Hybrid- ization-selection of mRNA with plasmids was performed as described (20). Twenty micrograms of each plasmid was 18S - linearized with HindIII and bound to 3-mm nitrocellulose 16S - squares. P. pyralis lantern poly(A)+ RNA (30 Ag) was hybridized to the filters in one tube at a final concentration of 200 1Lg of RNA/ml. Bound RNA was eluted from the filters by boiling. Nuclease-treated rabbit reticulocyte lysates were purchased from Bethesda Research Laboratories and were used according to the supplier's recommended conditions for optimal protein synthesis. One microgram of total lantern poly(A)+ RNA and 1/10th of the hybridization-selected RNAs each were translated in vitro in a final volume of20 FIG. 1. Blot hybridization analysis of firefly lantern poly(A)+ Aul. RNA. Total P. pyralis lantern poly(A)+ RNA (1 1Ag) was electro- Translation mixtures contained L-[35S]methionine (600 phoresed in each lane of a formaldehyde/1.3% agarose gel and then Ci/mmol, Amersham) at 2 mCi/ml. Five-microliter samples blotted onto a nitrocellulose filter. The individual lanes were cut of the translation products from hybridization-selected apart before hybridization. After hybridization to 32P-labeled DNA RNAs and a 1-1.l sample of the translated lantern poly(A)+ probe, the filters were autoradiographed. Ribosomal RNAs from RNA were analyzed by NaDodSO4/PAGE (21) followed by human (HeLa) and E. coli cells were run as size standards in an fluorography (22). Four microliters of each translation mix- adjacent lane, and their positions are indicated at left. Lane 1: ture was assayed for the presence of active firefly luciferase. Hybridization with LuclA cDNA, the 1200-bp EcoRI fragment Expression of Firefly Luciferase in E. coli. TB1, isolated from XLucl. Lane 2: hybridization of LuclB cDNA, the TB1(pKJB824.17), and TB1(pKW101) cells were grown in 10 270-bp EcoRI fragment isolated from XLucl. ml of Luria-Bertani medium (10 g of tryptone, 5 g of yeast extract, and 5 g of NaCl per liter, pH 7.4) at 30'C to OD650 = a single species of RNA that was 1.9-2.0 kb long; thus, both 0.5. The cells were heat-induced at 450C for 30 min and then of the EcoRI cDNA fragments in XLucl most likely resulted shifted to 370C for 1 hr. Cells were pelleted for 5 min at 3000 from the reverse transcription of a single species of mRNA. x g, and the supernatant was decanted. Pellets were resus- LuclA and LuclB Hybrid-Select Luciferase mRNA. The pended in 200 ,ul of 10 mM Tris Cl, pH 8.0/1 mM EcoRI cDNA fragments LuclA and LuclB were inserted into EDTA/lysozyme (1 mg/ml) and were incubated on ice for 10 the EcoRI site ofthe plasmid pUC13 to generate pLuclA and min. The cells were then frozen on dry ice and thawed. Fifty pLuclB, respectively. Plasmids pLuclA, pLuclB, and microliters of each cell lysate was assayed for luciferase pUC13 were used to hybrid-select mRNA from total firefly activity.
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