Departments of 1Chemical Engineering and 2Bioengineering, University of Washington, Box 351750, Seattle, WA 98195-1750, USA
3 To whom correspondence should be addressed. E-mail: baneyx{at}u.washington.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: biosensor/firefly/imaging/luc/luminescence/lux
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Luciferases oxidize chemically unrelated substrates in the presence of molecular oxygen to emit photons of visible light (Wilson and Hastings, 1998). The major advantage of these enzymes as reporters is the rapid (520 min) detection of metabolic events with high sensitivity and large dynamic range. Bacterial luciferase operons (e.g. the luxCDABE operons from Vibrio fischeri and Photorhabdus luminescens) which encode a heterodimeric luciferase (LuxAB) and all components necessary for light generation have been used in numerous biotechnology, environmental and genomic applications (Chatterjee and Meighen, 1995
; Rhodius et al., 2002
). Eukaryotic luciferases (e.g. the Photinus pyralis luc gene product) have the disadvantage of requiring exogenous addition of a luciferin substrate. However, they exhibit increased light production and higher efficiency than bacterial luciferases, exert a lower energy cost and metabolic burden on the host cell (Wilson and Hastings, 1998
), and are widely used for in vivo luminescence monitoring and for a variety of medical and pharmaceutical screens and applications (Doyle et al., 2004
; Roda et al., 2004
).
Luciferase from the North American firefly P.pyralis (EC 1.13.12.7) is a monooxygenase that catalyzes the ATP-dependent conversion of firefly luciferin into a luciferyl-adenylate, which is oxidized to electronically excited oxyluciferin in a multistep reaction (Ugarova, 1989). Relaxation to the ground state results in the production of yellowgreen light (
max
560 nm) with a remarkable quantum yield of 0.9 (Seliger and McElroy, 1960
; De Wet et al., 1985
). The 61 kDa enzyme consists of a large N-terminal domain (residues 1436) tethered to a smaller (residues 440550) C-terminal domain via a flexible linker in an anvil and hammer arrangement (Figure 1A) (Conti et al., 1996
). Formation of the active site likely requires interactions between the two structural domains and 15 residues have been proposed to be involved in the architecture of the luciferyl-adenylate binding pocket (dark space-filled residues in Figure 1A) (Branchini et al., 1998
, 2003
).
|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Plasmid pPluc*, which encodes the P.pyralis luc gene (Bonin et al., 1994), was obtained from the American Type Culture Collection (ATCC no. 87108). The random PCR mutagenesis protocol was adapted from Shafikhani et al. (1997)
. Primers 5'-AGGAAACAGACCATGGAAGACGCCA-3' and 5'-GTGGTGGTGGTGCTCGGTGCGGCCGCTCTAGAA-3' were used to amplify the 1652 bp luc gene from pPluc*, maintaining the NcoI site 2 bp upstream of the initiator codon and the NotI site downstream of the stop codon. PCRs consisted of 1x PCR buffer (Promega, Madison, WI, USA), 0.2 mM dATP, 0.2 mM dGTP, 1 mM dCTP, 1 mM dTTP, 1.25 mM MgCl2, 0.24 mM MnCl2, 0.1 µM of each primer, 1 µM (
500 ng) of pPluc* template and 5 U of Taq DNA polymerase (Promega) in a 100 µl final volume. The thermocycler was programmed for 30 cycles of 40 s at 90°C, 40 s at 50°C and 2 min at 72°C. PCR products were digested overnight with NcoI and NotI, and were gel purified.
Plasmid pTac24 was constructed by ligating a 482 bp SphIHindIII fragment from pKK233-2 (Amersham Pharmacia Biotech) into the same sites of pET24a (Novagen, Madison, WI, USA), thereby exchanging the T7 promoter for a trc promoter (Chow and Baneyx, 2005). This plasmid was digested with NcoI and NotI and the large DNA fragment was used as a backbone for ligation reactions with PCR-amplified products (typically 2 ng of backbone for 7 ng of amplified DNA in 10 µl final volume). Escherichia coli Top10 cells (Invitrogen, Carlsbad, CA, USA) were made electrocompetent and 50 µl were transformed with 3 µl of the ligation mixture. Aliquots (50 µl) were spread on LuriaBertani (LB) agar plates supplemented with 50 µg/ml neomycin. After overnight growth at 37°C, colonies were transferred onto nitrocellulose filters and luciferase synthesis was induced by spotting 1 mM IPTG on individual clones. After 1.5 h of incubation at 37°C, the filters were soaked with 1 mM D-luciferin (Biotium, Hollywood, CA, USA) in 100 mM sodium citrate buffer (pH = 5.0) for 15 min and colonies were examined in the dark for a change in bioluminescence color. Color-shifted mutants were streaked twice on LBneomycin plates and verified for altered luminescence before DNA sequencing of the full luc gene. Primers used for sequencing were 5'-CGTACCATGGAAGACGCCAAAAACATAAAG-3', which hybridizes at the start of luc; 5'-GATACTGCGATTTTAAGTGTTGTTCC-3', which hybridizes 700 bp downstream of the start of luc; 5'-CGGGCGTGGCAGGTCTTCCCGACGAT-3', which hybridizes 1400 bp downstream of the start of luc; and the reverse primer 5'-CGTATCTCTTCATAGCCTTATGCAG-3', which hybridizes 100 bp downstream of the start of luc. Approximately 10 000 colonies were screened. Plasmids encoding luciferases emitting orange and red light were named pTucO1, pTucO2, pTucR1 and pTucR2 (Table I).
|
The wild-type luc gene isolated from Ppluc* by NcoI/NotI digestion was subcloned into the same sites of pTac24 to yield pTuc. Site-directed mutagenesis was conducted on this plasmid using the QuikChange system (Stratagene, La Jolla, CA, USA) and primer pairs 5'-TCTAATTTACGCGAAATTGCTTCTGGGG-3' and 5'-CCCCAGAAGCAATTTCGCGCTAAATTAGA-3' for the H310R mutation, and 5'-CAAAGTGCGTTGATTGTACCAACCC-3' and 5'-GGGTTGGTACAATCAACGCACTTTG-3' for the L287I mutation. The presence of the correct mutations was verified by sequencing. The plasmid encoding the L287I mutation (C859A, A861T) was named pTucO2N and that containing the H310R mutation (A929G) was named pTucH310R. The L287I mutation was also introduced by site-directed mutagenesis into plasmid pTucO1 to combine the S293P and L287I substitutions. The resulting plasmid was named pTucGS.
Fluorescence spectroscopy
Shake flasks (125 ml) containing 25 ml of LB and 50 µg/ml neomycin were inoculated at a 1:50 dilution using overnight cultures of E.coli Top10 [F' endA1 recA1 hsdR17 (rK mK+) supE44 thi1 gyrA96 relA1
80
lacZ
M15
(lacZYA-argF) U169 deoR; Invitrogen] harboring pTuc, pTucO1, pTucO2, pTucR1, pTucR2, pTucO2N or pTucGS. Cells were grown at 30°C to A600
0.4, supplemented with 1 mM IPTG to induce luciferase expression and incubated for 1.5 h at the same temperature. Samples (1 ml) were collected and the A600 was adjusted to 0.9 using LB. Aliquots (1 ml) were centrifuged at 8000 g for 10 min, and cells were resuspended in 1 ml of 25 mM MES (pH = 5.5), 50 mM sodium phosphate (pH = 7.0) or 25 mM TrisHCl (pH = 8.0) containing 0.5 mM D-luciferin. The suspension was immediately transferred to a 5 ml fluorescence cuvette. After 5 min incubation at room temperature, emission spectra were collected on a Hitachi F-4500 fluorescence spectrophotometer operated in the luminescence mode. Data shown correspond to the average of 10 consecutive spectra collected within 15 min of substrate addition.
Immunoblotting
Cultures were grown and induced as above. Samples (3 ml) were harvested 1.5 h post-induction and the A600 was recorded. Cells were centrifuged at 8000 g for 8 min, resuspended in 3 ml of 50 mM potassium phosphate monobasic, pH 6.5, and disrupted with a French press at 10 000 psi. Soluble fractions were clarified by centrifugation at 8000 g for 20 min. Whole-cell samples were also collected 1.5 h post-induction, recovered by centrifugation at 8000 g for 2 min, resuspended in SDS loading buffer and disrupted by boiling. Soluble and insoluble cellular fractions corresponding to identical absorbance units were prepared as described previously (Thomas and Baneyx, 1996). Aliquots were resolved on 12.5% SDS minigels and proteins were transferred onto nitrocellulose. Membranes were probed with polyclonal goat anti-luciferase antibodies (Promega) at a 1:1000 dilution followed by incubation with alkaline phosphatase-conjugated rabbit anti-goat IgG at 1:1000 dilution. Blots were developed using 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium.
![]() |
Results and discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In an effort to select luciferase variants displaying altered bioluminescence colors, we subjected the P.pyralis luc gene to random PCR mutagenesis as described in Materials and methods. Approximately 10 000 colonies were screened. Promising clones were identified by unaided visual examination to guarantee that light production would be sufficient for in vivo applications. Four bright variants emitting in the orange to red regions of the spectrum were selected for further analysis (Table I).
Sequencing of the mutant genes revealed that a transition leading to a single amino acid substitution was responsible for the color change in variants LucO1, LucR1 and LucR2. Variant LucO2 contained two substitutions resulting from both a transition and a transversion. All mutations mapped to the ß-sheet B subdomain of the large N-terminal portion of luciferase, which involves residues 2270 and 236351 and consists of six ß-strands and six -helices (Conti et al., 1996
). Remarkably, all substitutions were clustered in the region encompassing ß-strand B5, helix 10 and ß-strand B6, and none was located in the predicted luciferin-binding site, which involves 13 amino acids from the B subdomain (Figure 1A) (Branchini et al., 1998
). Possible explanations for this result are that mutagenesis was not carried out to saturation and/or the fact that mutations in active site residues often decrease luciferase-specific activity (Branchini et al., 1998
, 1999
, 2003
), which could preclude visual detection of color-shifted variants. Considering that the B5
10B6 region directly connects to the Ser314Leu319 loop that is a mobile component of the putative active site (Franks et al., 1998
), the mutations described here may indirectly alter luminescence color by modulating loop location (Figure 1B). The fact that the G315A and G316A substitutions red-shift the maximum emission wavelength (
max) of P.pyralis luciferase (Branchini et al., 2003
) is consistent with this interpretation.
Expression, in vivo folding and proteolytic stability
To assess the impact of the mutations on luciferase structural integrity and in vivo folding, cultures expressing wild-type and mutant proteins were grown in LB medium at 30 or 37°C. Samples collected 90 min post-induction were separated into whole-cell, soluble and insoluble fractions and subjected to western analysis with anti-luciferase antiserum. Figure 2 shows that all strains produced comparable amounts of luciferase at both temperatures and that soluble cell fractions contained two immunoreactive bands corresponding to full-length luciferase and to a 59 kDa product (lanes s). Since the truncated fragment was not present in whole-cell fractions (lanes w), it probably results from a proteolytic event during sample processing. We surmised that the culprit was OmpT, an outer membrane protease specific for paired basic residues (Sugimura and Nishihara, 1988
) that is well known for cleaving intracellular proteins following cell disruption (Baneyx and Mujacic, 2004
). Indeed, when plasmid pLuc was introduced into the ompT deletion strain SF100 (Baneyx and Georgiou, 1990
), the
59 kDa band disappeared, while it remained present in isogenic ompT+ cells (data not shown). The existence of a degradation fragment (which is consistent with OmpT-mediated cleavage of the K510K511 bond) provided us with an opportunity to assess how our mutations affected the structural integrity of luciferase since destabilized mutants are usually more sensitive to proteolytic attack than properly folded proteins.
|
Spectral characteristics
Because the intended applications of the luciferase mutants described herein are in the biosensing and imaging fields, we collected bioluminescence emission spectra using whole-cell samples from cultures grown at 30°C. At neutral pH, cells synthesizing the LucR1 or LucR2 variants exhibited a single emission peak with max shifted to the 600610 nm range and showed no significant peak broadening relative to authentic luciferase (Figure 3 and Table II). However, the orange color of cultures expressing the LucO1 and LucO2 mutants was due to a major red-shifted peak with
max of
600 nm and a minor peak with maximum emission wavelength near that of the native protein (Figure 3 and Table II).
|
|
Site-directed mutagenesis
To dissect the contribution of the two substitutions in the LucO2 variant, the L287I and H310R mutations were separately introduced in the native luciferase gene by site-directed mutagenesis, yielding LucO2N and LucH310R, respectively. Inspection of bioluminescence spectra collected as above revealed that the L287I substitution was predominantly responsible for the color change despite the fact that the side chain of Leu287 projects away from the putative active site (Figures 1B and 4; Table II). Thus, just like in the case of L.cruciata luciferase (Kajiyama and Nakano, 1991), a single amino acid change is sufficient to cause a pronounced alteration in the color of the light emitted by all our variants of P.pyralis luciferase.
|
Influence of pH on light emission
It has long been known that the firefly luciferase emission spectrum becomes red-shifted when the enzyme is assayed at low pH (DeLuca, 1976). Because the internal pH of E.coli varies following acid or alkaline shock (Zilberstein et al., 1984
; Richard and Foster, 2004
), we next examined how basic and acidic environments affected the spectral characteristics of luciferase-producing cultures.
Results obtained with cells producing native luciferase mirrored those reported with the purified enzyme (Branchini et al., 1999). Although an increase in pH from 7 to 8 had little impact on spectral characteristics, acidification of medium led to a 50 nm red-shift in
max (Figure 5 and Table II). In fact, the majority of our mutants exhibited narrow bandwith emission spectra with maximum emission wavelengths in the 610 nm range when cells were assayed at low pH. One exception was the LucGS variant, which displayed a bimodal spectrum at pH 5.5 with a minor peak of
max = 556 nm about half as intense as the major red-shifted peak.
|
The fact that all mutants primarily luminesce in the red upon acid shock and in the yellowgreen upon alkaline shock indicates that the internal pH of E.coli is very similar to the extracellular pH shortly after imposition of pH stress. It is also worth noting that, for all variants, including LucGS, we were able to identify pH conditions leading to a distinct bimodal emission spectrum. These results support the proposal that firefly luciferase bioluminescence color is determined by the relative contribution of short- and long-wavelength emission peaks, each of which corresponds to the preferential stabilization of a different resonance species of excited oxyluciferin that is ultimately controlled by the protein structure (Branchini et al., 2004).
![]() |
Conclusions |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Baneyx,F. and Mujacic,M. (2004) Nat. Biotechnol., 22, 13991408.[CrossRef][ISI][Medline]
Bonin,A.L., Gossen,M. and Bujard,H. (1994) Gene, 141, 7577.[CrossRef][ISI][Medline]
Branchini,B.R., Magyar,R.A., Murtiashaw,M.H., Anderson,S.M. and Zimmer,M. (1998) Biochemistry, 37, 1531115319.[CrossRef][ISI][Medline]
Branchini,B.R., Magyar,R.A., Murtiashaw,M.H., Anderson,S.M., Helgerson, L.C. and Zimmer,M. (1999) Biochemistry, 38, 1322313230.[CrossRef][ISI][Medline]
Branchini,B.R., Magyar,R.A., Murtiashaw,M.H. and Portier,N.C. (2001) Biochemistry, 40, 24102418.[CrossRef][ISI][Medline]
Branchini,B.R., Southworth,T.L., Murtiashaw,M.H., Boije,H. and Fleet,S.E. (2003) Biochemistry, 42, 1042910436.[CrossRef][ISI][Medline]
Branchini,B.R., Southworth,T.L., Murtiashaw,M.H., Magyar,R.A., Gonzalez, S.A., Ruggiero,M.C. and Stroh,J.G. (2004) Biochemistry, 43, 72557262.[CrossRef][ISI][Medline]
Chatterjee,J. and Meighen,E.A. (1995) Photochem. Photobiol., 62, 641650.[ISI]
Chow,I.-T. and Baneyx,F. (2005) FEBS Lett., 579, 42354241.[CrossRef][ISI][Medline]
Conti,E., Franks,N.P. and Brick,P. (1996) Structure, 4, 287298.[CrossRef][ISI][Medline]
De Wet,J.R., Wood,K.V., Helinski,D.R. and DeLuca,M. (1985) Proc. Natl Acad. Sci. USA, 82, 78707873.
DeLuca,M. (1976) Adv. Enzymol., 44, 3768.[ISI][Medline]
Doyle,T.C., Burns,S.M. and Contag,C.H. (2004) Cell Microbiol., 6, 303317.[CrossRef][ISI][Medline]
Franks,N.P., Jenkins,A., Conti,E., Lieb,W.R. and Brick,P. (1998) Biophys. J., 75, 22052211.
Greer,L.F.,III and Szalay,A.A. (2002) Luminescence, 17, 4374.[CrossRef][ISI][Medline]
Guex,N. and Peitsch,M.C. (1997) Electrophoresis, 18, 27142723.[ISI][Medline]
Kajiyama,N. and Nakano,E. (1991) Protein Eng., 4, 691693.[ISI][Medline]
Kneen,M., Farinas,J., Li,Y. and Verkman,A.S. (1998) Biophys. J., 74, 15911599.
Rhodius,V., Van Dyk,T.K., Gross,C. and LaRossa,R.A. (2002) Annu. Rev. Microbiol., 56, 599624.[CrossRef][ISI][Medline]
Richard,H. and Foster,J.W. (2004) J. Bacteriol., 186, 60326041.
Roda,A., Pasini,P., Mirasoli,M., Michelini,E. and Guardigli,M. (2004) Trends Biotechnol., 22, 295303.[CrossRef][ISI][Medline]
Seliger,H.H. and McElroy,W.D. (1960) Arch. Biochem. Biophys., 88, 136141.[CrossRef][ISI][Medline]
Shafikhani,S., Siegel,R.A., Ferrari,E. and Schellenberger,V. (1997) Biotechniques, 23, 304310.[ISI][Medline]
Sugimura,K. and Nishihara,T. (1988) J. Bacteriol., 170, 56255632.[ISI][Medline]
Thomas,J.G. and Baneyx,F. (1996) J. Biol. Chem., 271, 1114111147.
Ugarova,N.N. (1989) J. Biolumin. Chemilumin., 4, 406418.[CrossRef][ISI][Medline]
Visick,K.L. and McFall-Ngai,M.J. (2000) J. Bacteriol., 182, 17791787.
Wilson,T. and Hastings,J.W. (1998) Annu. Rev. Cell Dev. Biol., 14, 197230.[CrossRef][ISI][Medline]
Zilberstein,D., Agmon,V., Schuldiner,S. and Padan,E. (1984) J. Bacteriol., 158, 246252.[ISI][Medline]
Received June 1, 2005; revised August 22, 2005; accepted September 12, 2005.
Edited by Ashutosh Chilkoti
|