Biochimica et Biophysica A cta, 1131 (1992) 161-165 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4781/92/$05.00

161

BBAEXP 92387

Molecular cloning and expression in Escherichia coli of a c D N A clone encoding luciferase of a firefly, Luciola lateralis Hiroki Tatsumi, Naoki Kajiyama and Eiichi Nakano Research and Development Ditqsion, Kikkoman Corporation, Noda (Japan)

(Received 4 January 1992)

Key words: Firefly luciferase; Recombinant DNA; Primary structure; (L. cruciata); (P. pyralis)

We have cloned a cDNA encoding Luciola lateralis (a common firefly in Japan) luciferase from a cDNA library of lantern poly(A) + RNA, using a cDNA of L. cruciata (another common firefly in Japan) luciferase as a probe. The primary structure of L. lateralis luciferase deduced from the nucleotide sequence was shown to consist of 548 amino acids with a molecular weight of 60132. Sequence comparison indicates that L. lateralis luciferase has significant,sequence identity (94%) to L. cruciata luciferase, and that it has less sequence similarity (67%) to Photinus pyralis (a North American firefly) luciferase. The isolated cDNA clone, when introduced into Escherichia coli, directed the synthesis of enzymatically active luciferase under the control of the lacZ promoter.

Introduction

Firefly luciferase catalyzes the oxidation of luciferin in the presence of ATP, 0 2 and Mg 2÷, producing light [1]. The luciferase of Photinus pyralis (a North American firefly) has been purified and extensively characterized [1]. Recently, we have purified luciferases from two fireflies commonly observed in Japan, Luciola cruciata and Luciola lateralis, and found that L. lateralis luciferase (LIL) has some unique properties in comparison with L. cruciata luciferase (LcL) and P. pyralis luciferase (PpL) [2]. LIL is superior to LcL and PpL in thermal and pH stabilities, and the reaction catalyzed by LIL emits light with a peak intensity at 552 nm, which is 10 nm shorter in wavelength than those of LcL and PpL. cDNAs for LcL and PpL have already been isolated and their amino acid sequences have been deduced from the nucleotide sequences [3-5]. To understand the difference in properties of these luciferases at molecular level, we attempted to isolate a cDNA clone encoding L1L.

Abbreviations: IPTG, isopropyl-/3-thiogalactoside; LcL, luciferase of Luciola cruciata; LIL, luciferase of Luciola lateralis; ORF, open reading frame; PpL, luciferase of Photinus pyralis; Amax, the wavelength of maximum intensity of light. Correspondence: H. Tatsumi, Research and Development Division, Kikkoman Corporation, 399 Noda, Noda city, Chiba 278, Japan.

In this paper, we describe the cloning and sequence analysis of LIL cDNA. Sequence comparison reveals that L1L is more similar to LcL than it is to PpL, although LIL has some unique properties among these three luciferases. We also describe the expression of L1L cDNA in Escherichia coli. Materials and Methods

Strains, media and plasmids. Escherichia coli strain -- + DH1 ( F - recA1 endA1 gyrA96 thi-1 hsdR17 (rkm k) supE44) was used as a carrier for recombinant plasmids. E. coli strain JM101 (A(lac-pro) supE:thi (F' traD36 proAB ÷ laclqZAM15)) was used for the DNA sequencing and the expression of L1L cDNA. E. coli ceils were grown in L broth (1% Bacto tryptone, 0.5% yeast extract, 0.5% NaC1). When necessary, 50 ~g ampicillin/ml was added. Various pUC plasmids were purchased from Takara Shuzo (Kyoto). Materials. Live fireflies were purchased from the Seibu department store, Ikebukuro branch (Tokyo). All restriction enzymes and T4 DNA ligase were obtained from Takara Shuzo, and used as recommended by the supplier. Purified PpL was purchased from Sigma. Culture, extraction and assay of luciferase expressed in E. coli. JM101 (pHLf7) or JM101 ( p U C l l 9 ) cells were cultured in 3 ml of L broth at 37°C with shaking at 120 rpm for 18 h. The cultures, 0.5 ml each, were inoculated in 10 ml of L broth with or without the addition of 1 mM IPTG. After growth at 37°C with shaking at

162 120 rpm for 4 h, the culture were subjected to centrifu g a t i o n to give 20 mg of cell pellets. Extraction and assay of luciferase p r o d u c e d in E. coli were carried out as described by de W e t et al. [6] with some modifications. E. coli pellets (20 mg) were r e s u s p e n d e d in 1 ml of lysis buffer (100 m M K P O 4 (pH 7.8), 2 m M E D T A , 1 m M dithiothreitol, 0.1% lysozyme, 0.02% p r o t a m i n e sulfate), i n c u b a t e d on ice for 15 rain, and then frozen on dry ice. T h e frozen pellets were allowed to thaw at 25°C and cleared by c e n t r i f u g a t i o n for 5 min. T h e n , 10 ~I of the extract was a d d e d to 400 ~1 of substrate mix (25 m M glycylglycine ( p H 7.8), 5.4 m M MgSO4, 0.086 m M luciferin). Each test t u b e was placed in a L u m i n e s c e n c e R e a d e r BLR-201 (Aloka, Tokyo), a n d 100 /~1 of A T P solution (20 m M A T P , 25 m M glycylglycine (pH 7.8)) was injected. T h e c o u n t which is p r o p o r t i o n a l to the n u m b e r of p h o t o n s emitted was i n t e g r a t e d for 20 s.

Isolation o f poly(A) + R N A from L. lateralis lanterns. L a n t e r n s were dissected out from 5 g of frozen L. lateralis fireflies and a d d e d to 18 ml of g u a n i d i n e isothiocyanate solution (6 M g u a n i d i n e isothiocyanate, 37.5 m M sodium citrate (pH 7.0), 0.75% sodium Nlauroylsarcosine, 0.15 M / 3 - m e r c a p t o e t h a n o l ) f o l l o w e d by h o m o g e n i z a t i o n with a h o m o g e n i z e r ( N i h o n Seiki, Tokyo; Ace h o m o g e n i z e r AM-3). T h e h o m o g e n a t e was t h e n subjected to s e d i m e n t a t i o n t h r o u g h a CsC1 cushion a n d 340 /zg of total R N A was extracted as described previously [7]. Finally, 6 kLg of poly(A) + R N A was isolated using an oligo(dT)-cellulose column. Construction o f c D N A library. F r o m 2 ~ g of the l a n t e r n poly(A) + R N A , 0.25 /~g of d o u b l e s t r a n d e d c D N A was synthesized using a c D N A synthesis kit ( A m e r s h a m ) . A f t e r E c o R I sites of the c D N A were mhethylated and E c o R I linkers ( G G A A T T C C ) were a d d e d to both ends of the c D N A by the use of c D N A

TABLE 1

Codon usage for luciferase cDNAs from Luciola lateralis (LIL), L. cruciata (LcL) and Photinus pyrafis (PpL) LIL

LcL

PpL

LIL

LcL

PpL

TTT Phe TTC Phe TTA Leu TI'G Leu

19 4 20 11

18 5 17 9

18 12 11 14

TCT Ser TCC Ser TCA Ser TCG Ser

12 1 7 1

13 2 5 1

9 7 2 5

TAT Tyr TAC Tyr TAA Stop TAG Stop

14 8 0 0

13 8 0 0

8 11 0 0

TGT Cys TGC Cys TGA Stop TGG Trp

5 2 0 1

7 1 0 1

2 2 0 2

CTT Leu CTC Leu CTA Leu CTG Leu

11 2 3 2

12 2 4 5

8 5 5 9

CCT Pro CCC Pro CCA Pro CCG Pro

8 0 15 5

10 2 13 4

8 7 7 7

CAT His CAC His CAA Gin CAG Gin

5 3 12 0

5 3 12 1

8 6 9 7

CGT Arg CGC Arg CGA Arg CGG Arg

9 2 1 0

9 2 2 0

2 3 4 1

ATT lie ATC lie ATA Ile ATG Met

23 6 7 12

22 5 6 11

18 11 9 14

ACT Thr ACC Thr ACA Thr ACG Thr

19 4 7 4

14 8 14 0

7 7 9 6

AAT Asn AAC Asn AAA Lys AAG Lys

10 10 37 7

12 8 40 3

9 10 26 14

AGT Ser AGC Ser AGA Arg AGG Arg

6 2 9 0

7 2 8 0

5 1 10 0

GTT Val GTC Val GTA Val GTG Val

23 5 14 7

33 4 15 3

17 9 7 11

GCT Ala GCC Ala GCA Ala GCG Ala

17 3 13 1

12 6 13 1

8 13 8 13

GAT Asp GAC Asp GAA Glu GAG Glu

22 5 34 6

20 5 33 6

17 14 23 10

GGT Gly GGC Gly GGA Gly GGG Gly

27 10 14 1

23 9 18 1

8 9 19 9

163 cloning kit (Amersham), the cDNA was inserted in the EcoRI site of pUCll9, and transformed into E. coli strain DH1, giving a cDNA library consisting of about 10 000 independent recombinant clones. Nucleotide sequencing procedure. Nucleotide sequencing was carried out basically by the method of Henikoff [8]. The 1.5 kb and 0.3 kb EcoRI fragments cloned in pHLf7 were, respectively, inserted into the EcoRI site of pUCll8. The resultant plasmids were digested with KpnI +BamHI, and subjected to deletion at approx, every 150 bp in the insert by the use of a deletion kit for kilo base sequences (Takara Shuzo). Deleted plasmids at approx, every 150 bp in the insert were selected. Single-stranded plasmids were prepared using a helper phage, M13KO7 (Takara Shuzo). The sequence of the cDNA fragment in every plasmid was determined by the dideoxy chain-termination method. Both strands were determined by overlapping at the junction of each deleted fragment. Results and Discussion

Isolation of a LlL cDNA clone Using the 2.0 kb PstI fragment of the previously cloned LcL cDNA [3] as a DNA probe, LIL cDNA was screened among the cDNA library of L. lateralis lantern poly(A) + RNA by the colony hybridization method [9]. Several clones were found to be positive in this selection, and the plasmid with the longest insert (1.8 kb) was designated as pHLf7 (Fig. 1) and used for subsequent experiments. Sequence analysis of LlL cDNA The nucleotide sequence of L1L cDNA cloned on pHLf7 was determined (Fig. 2). The cDNA is found to be 1781 bp long with a continuous open reading frame (ORF) of 1644 bp. The ORF encodes a protein consisting of 548 amino acids with a calculated molecular weight of 60 132. The 5' noncoding region is 58 bp long

LcL cDNA c~

.@ et

lacP

pHLf7

Ap Fig. 1. Structure of pHLfT. Arrow shows the direction of transcription. Abbreviations: Ap, ampicillin; lacP, lacZ promoter; RI, EcoRI; RV, EcoRV; S, Sspl.

and the Y noncoding region is 79 bp long. The 3' noncoding region seems to be truncated, since it lacks a poly(A) tail at the terminus. There are two ATG triplets at nucleotide positions 59 and 68 in the 5' terminus of the ORF. In 95% of all cases, the ATG triplet nearest to the 5' end of the mRNA is the initiator signal [10], and four stop codons (nucleotide positions 11, 38, 41 and 53) lie just upstream from the ORF. These findings suggest that the ATG at nucleotide position 59 is the codon that initiates translation. The nucleotide sequences of LcL cDNA [3] and PpL cDNA [5] have already been determined. The AT content in the ORF of L1L cDNA (63%) and LcL cDNA (63%) is relatively high compared with that of PpL cDNA (55%). With regard to the codon distribution, L1L cDNA is quite similar to LcL cDNA, but PpL cDNA have a different codon selection, mainly because L1L and LcL DNAs prefer A and T at the third codon (Table I). According to the deduced amino acid sequences, these three luciferases are similar in size. LIL, LcL and PpL consist of 548, 548 and 550 amino acids, respectively. Fig. 3 shows the alignment of the primary structures of L1L, LcL and PpL. L1L showed significant similarity to LcL throughout the sequence (the overall identity is 94%). On the contrary, many amino acid substitutions are observed between L1L and PpL, especially in the first 200 amino acids from the N-terminus (the overall identity is 67%). These observations are taxonomically reasonable, since L. lateralis and L. cruciata belong to the same genus, whereas P. pyralis belongs to a different one. Although there are strong sequence similarities, it is interesting to note that some enzymatic properties of L1L are different from those of LcL and PpL [2]. LcL and PpL are found to have similar thermal and pH stabilities, whereas L1L is more stable at high temperature (when tested at 50°C for 30 min, the remaining activity of L1L is around 20%, while LcL and PpL are completely inactivated) and at a wide pH range (when incubated at 4°C for 4 h, L1L retains approx. 50% of its activity at pH 5.5 and 11.0, but the remaining activities of LcL and PpL are less than 20% at these pH values). Moreover, )tmax produced by purified L1L is 552 nm, which is 10 nm shorter than those of LcL and PpL. This may be because some limited residues which are the same in LcL and PpL but different in L1L are crucial for stability and Areax. In the case of LcL, some single amino acid substitutions are shown to cause drastic shifts of /~max (up to 50 nm) by means of random mutagenesis [11]. In the study of the luciferase of a click beetle, Pyrophorus plagiophthalamus, two pairs of amino acid substitution are demonstrated to be responsible for the difference of Areax by constructing hybrid proteins [12]. At present, we can not identify the crucial residues for stability and Areax. Further studies

~..

_~.

g

g

='Z

.~

O0

>

i-~

0

0

O~

I.~

-4

c1

.--]

U~

.

~>

~ 0

~

0 0

~

~

~

~

~

~ 0

>

>

~

~

~ 0

~

~ 0

>

>

~

~>

~ 0

~

~

=>

~

~ 0

~

>

~

~>

~

~ 0

~

~

~

~>

~ 0

~

~

~

~ 0

>

>

~

~

>

0 0

~

~

~>

~

~ 0

>

>

~>

~ 0

>

~

~

~>

~

~ 0

>

~

~>

0

>

>

165

LIL LcL PpL

MENMENDENIVYGPEPFYPI EEGSAGAQLRKYMDRYA-KLGAI AFTNALTGVDYTYAEYL V K T E V S --DAK KK A L D T E H A K LVP T D H I E NI F

59

Expression of Luciola lateralis luci~erase in Escherichia coli

EKSCCLGEALKNYGLWDGRIALCSENCEEFFIPVLAGLFIGVGVAPTNEIYTLRELVHS 119 K Q I M VR A M R NTNH W SLQ M GA A A D NE LN LGI S K P T I V F S S K K G L D K V I T V Q K T V T A I K T I V I L D S K V D Y R G Y Q S M D N F I K K N T P Q G F K 179 T CL T R P Q MN Q V V Q ILN K L P I QK I M T Q F YT VTSHL P N GSSFKTVEVNRKEQVAL IMNSSGSTGLPKGVQLTHENAVTRFSHARDP IYGNQVSPGTAI 239 A D T V E Y D V P E S F D DKTI A P R T CV F IID LTWpFHH S

G F G M F T T L G Y L T C G F R I V M L T K F D E E T F L K T L Q D Y K C S SV I L V P T L F A I LNR 999 I V T K I V LMYR E L RS IQ AL SFFAK

SELLDKYDLSNLVEIASGGAPLSKEI GEAVARRFNLPGVRQGYGLTETTSAI N V T I H V K H I

I ITPEGDD

S

A w

DE

359

419

K Y K G Y Q V P P A E L E S V L L Q H P N I F D A G V A G V P D P IA 479 S V A I L DD

GELPGAVVVLEKGKSMTEKEVMDYVASQVSNAKRLRGGVRFVDEVPKGLTGKIDGKAIRE S N R A H T IV TT K V L ARK

539

I L K K P V A K M ...... I --

Host (plasmid)

IPTG

Count a

JM101 (pHLf7) JM101 (pHLf7) JM101 (pUCI19) purified PpL

+ -

6.2-106 2.3.105 < 1.0.104 2.4.106

For the first three lines, count/ml of culture/20 s. For the last line (control), count/1.0 gg of purified Photinus pyralis luciferase/20 S.

L

KPGASGKVVPLFKAKVIDLDTKKTLGPNRRGEVCVKGPMLMKGYVDNPEATREI IDEEGW s N K L V F E V G V Q L R I S N NAL KD LHTGDI GYYDEEKHFFIVDRLKSLI

TABLE II

KGGKSKL

Fig. 3. Comparison of amino acid sequences of Luciola lateralis luciferase (LIL), L. cruciata luciferase (LcL) and Photinus pyralis luciferase (PpL). For LcL and PpL, residues which are different from the corresponding LIL residues are indicated. The numbering is that of LIL. Gaps (-) were introduced to optimize the amino acid alignment. with s i t e - d i r e c t e d m u t a g e n e s i s m i g h t b e able to elucid a t e such residues. A l t e r n a t i v e l y , a c o n f o r m a t i o n a l c h a n g e o f e n z y m e s might c o n t r i b u t e to t h e d i f f e r e n c e in p r o p e r t i e s r a t h e r t h a n specific residues. T h e solution of crystal s t r u c t u r e could clarify this question.

Expression o f LlL c D N A in E. coli T h e lacZ p r o m o t e r on p H L f 7 is l o c a t e d just ups t r e a m o f the LIL c D N A , a n d t r a n s c r i p t i o n of these two e l e m e n t s a r e in the s a m e d i r e c t i o n (Fig. 1). Thus, we t h o u g h t p H L f 7 c o u l d d i r e c t the synthesis o f LIL in E. coli, a n d p e r f o r m e d t h e following e x p e r i m e n t . R e c o m b i n a n t E. coli cells, JM101 ( p H L f 7 ) a n d JM101 ( p U C l l 9 ) , w e r e c u l t u r e d with o r w i t h o u t t h e a d d i t i o n of 1 m M I P T G , an i n d u c e r of the lacZ p r o m o t e r , a n d cell extracts as well as a p u r i f i e d P p L as a c o n t r o l w e r e t e s t e d for luciferase activities. A s shown in T a b l e II, a luciferase activity was d e t e c t e d in an extract f r o m JM101 ( p H L f 7 ) c u l t u r e d with t h e a d d i t i o n of 1 m M I P T G , a n d a low activity was d e t e c t e d in an extract of JM101 ( p H L f 7 ) c u l t u r e d w i t h o u t the a d d i -

tion o f I P T G . N o activity was d e t e c t e d in extracts f r o m JM101 ( p U C l l 9 ) . T h e s e results d e m o n s t r a t e t h a t the L1L c D N A on p H L f 7 was e x p r e s s e d u n d e r the c o n t r o l o f the lacZ p r o m o t e r w i t h o u t t h e a r r a n g e m e n t o f t h e n u c l e o t i d e s e q u e n c e b e t w e e n the p r o m o t e r a n d the start codon. T h e a m o u n t of LIL p r o t e i n p r o d u c e d in E. coli with the a d d i t i o n o f 1PTG was e s t i m a t e d to be a r o u n d 2.6 / z g / m l , since the specific activities of rec o m b i n a n t L1L p r o d u c e d in E, coli a n d P p L from Photinus pyralis w e r e a l m o s t the s a m e ( u n p u b l i s h e d result).

Acknowledgement W e t h a n k Mrs. K a z u k o S a i t o h for h e r technical assistance.

References 1 DeLuca, M. and McElroy, W.D. (1978) Methods Enzymol. 57, 3-15. 2 Kajiyama, N., Masuda, T., Tatsumi, H. and Nakano, E. (1992) Biochim. Biophys. Acta, in press. 3 Masuda, T., Tatsumi, H. and Nakano, E. (1989) Gene 77,265-270. 4 De Wet, J.R., Wood, K.V., Helinski, D.R. and DeLuca, M. (1985) Proc, Natl. Acad. Sci. USA 82, 7870-7873. 5 De Wet, J.R., Wood, K.V., DeLuca, M., Helinski, D.R. and Subramani, S. (1987) Mol. Cell. Biol. 7, 725-737. 6 De Wet, J.R., Wood, K.V., Helinski, D.R. and DeLuca, M. (1986) Methods Enzymol. 133, 3-14. 7 Tatsumi, H., Masuda, T. and Nakano, E. (1988) Agric. Biol. Chem. 52, 1123-1127. 8 Henikoff, S. (1984) Gene 28, 351-359. 9 Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning, A Laboratory Manual, pp. 312-328, Cold Spring Harbor Laboratory, Cold Spring Harbor. 10 Kozak, M. (1984) Nucleic Acids Res. 12, 857-872. 11 Kajiyama, N. and Nakano, E. (1991) Protein Eng. 4, 691-693. 12 Wood, K.V., Lam, Y.A., McElroy, W.D. and Seliger, H.H. (1989) J. Biolumin. Chemilumin. 4, 31-39.