Comparison of enhanced bioluminescence energy transfer donors for protease biosensors

Analytical Biochemistry 424 (2012) 206–210

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Comparison of enhanced bioluminescence energy transfer donors for protease biosensors Helen Dacres ⇑, Michelle Michie, Stephen C. Trowell CSIRO Food Futures National Research Flagship & CSIRO Ecosystem Sciences, Australia, P.O. Box 1700, Canberra, ACT 2601, Australia

a r t i c l e

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Article history: Received 16 December 2011 Received in revised form 17 February 2012 Accepted 22 February 2012 Available online 1 March 2012 Keywords: Bioluminescence Coelenterazine RLuc2 RLuc8 Thrombin Protein–protein

a b s t r a c t Bioluminescence energy transfer (BRET) is a powerful tool for the study of protein–protein interactions and conformational changes within proteins. We directly compared two recently developed variants of Renilla luciferase (RLuc), RLuc2 and RLuc8, as BRET donors using an in vitro thrombin assay. The comparison was carried out by placing a thrombin-specific cleavage sequence between the donor luciferase and a green fluorescent protein (GFP2) acceptor. Substitution of native RLuc with the RLuc mutants, RLuc2 and 8, in a BRET2 fusion protein increased the light output by a factor of 10. Substitution of native RLuc with either of the RLuc mutants resulted in a decrease in BRET2 ratio by a factor of 2 when BRET2 components were separated by the thrombin cleavage sequence. BRET2 ratios changed by factors of 18.8 ± 1.2 and 18.2 ± 0.4 for GFP2-RG-RLuc2 and GFP2-RG-RLuc8 fusion proteins, respectively, on thrombin cleavage compared to 28.8 ± 0.20 for GFP2-RG-RLuc. The detection limits for thrombin were 0.23 and 0.26 nM for RLuc2 and RLuc8 BRET2 systems, respectively, and 15 pM for GFP2-RG-RLuc. However, overall, the mutant BRET systems remain more sensitive than FRET and brighter than standard BRET2. Crown Copyright Ó 2012 Published by Elsevier Inc. All rights reserved.

Bioluminescence resonance energy transfer (BRET)1 is a naturally occurring phenomenon found in a variety of marine organisms such as Aequorea victoria and Renilla reniformis and results from the nonradiative transfer of energy from a bioluminescent donor, Renilla luciferase (RLuc) to a fluorescent acceptor green fluorescent protein (GFP) [1]. Over the last decade, the BRET approach has been used extensively for monitoring protein–protein interactions in living cells, especially homo- and hetero-dimerization of G-protein coupled receptors (GPCRs) [2]. It has also been used for monitoring GPCR interactions with G-proteins [3] and b-arrestin [4,5] and interactions between other proteins involved in GPCR function and regulation [6]. We recently demonstrated that BRET systems are more sensitive than FRET for monitoring protease cleavage [7,8] and that the optimal working distance of the BRET2 system is significantly larger than for FRET [9] making it more suitable for monitoring conformational changes in GPCRs [10]. The BRET2 system uses RLuc with the substrate coelenterazine 400a (Clz400a) as the energy donor and a green fluorescent acceptor protein (GFP2) acceptor. Unfortunately, the advantages associated with the BRET2 system are partly outweighed by the low quantum yield and the rapid decay kinetics of the Clz400a donor substrate. It was recently

⇑ Corresponding author. Fax: +61 2 6246 4094. E-mail address: [email protected] (H. Dacres). Abbreviations used: BRET, bioluminescence resonance energy transfer; GFP, green fluorescent protein; GPCRs, G-protein coupled receptors; RLuc, Renilla luciferase; SDSPAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis. 1

shown that the sensitivity of the BRET2 assay can be significantly improved by substituting native RLuc with mutant RLuc donors with improved quantum yield and stability [11,12]. Substituting RLuc8 for native RLuc in a BRET2 fusion protein increased the BRET ratio by a factor of 5.5, thereby potentially enhancing the dynamic range of the BRET2 system [12]. Improvements in the sensitivity of BRET assays for the detection of ligand-induced GPCR–b-arrestin interactions in live cells were also demonstrated [4,5]. Due to these properties, RLuc variants, particularly RLuc8, are increasingly the preferred luciferase donors for BRET-based reporter systems [5,12–16]. It is now generally accepted that substitution of native RLuc with RLuc2 or RLuc8 as the luciferase donor will improve the performance characteristics of the BRET2 assay but this has never been directly assessed in vitro. Therefore we used a wellestablished in vitro approach to test this assumption. A thrombin-specific cleavage sequence was inserted between the donor and the acceptor proteins allowing thrombin cleavage to be detected through changes in the BRET2 signal. Thrombin is involved in the regulation of blood coagulation [17], selectively cleaving the Arg–Gly bonds of fibrinogen to form fibrin. The RLuc8 donor has been used as a BRET donor when coupled to quantum dot acceptors for the detection of matrix metalloproteinase (MMP) protease activity [13,16] but RLuc2 performance has not been assessed using any protease-based biosensor. The results of this study demonstrate that the increase in luminescence afforded to the BRET2 system by substituting RLuc with RLuc mutants comes at the expense of assay sensitivity.

0003-2697/$ - see front matter Crown Copyright Ó 2012 Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ab.2012.02.028

Enhanced protease biosensor / H. Dacres et al. / Anal. Biochem. 424 (2012) 206–210

Materials and methods Materials All primers (Table S1, Supplementary Material) were purchased from Geneworks (Australia). RLuc2 and RLuc8 cDNAs were amplified from constructs pcDNA3.1-RLucC124A/M185V (RLuc2) and pcDNA3-RLuc8 and kindly provided by Sanjiv Gambhir (Stanford University, CA, USA). Construction of BRET proteins RLuc2 and RLuc8 were amplified by polymerase chain reaction (PCR) using the primers shown in Table S1 (Supplementary Material) and cloned into pGEM-T Easy vector (Promega, Australia). This resulted in the insertion of PstI and the sequence encoding the thrombin recognition site (LQGSLVPRGSLQ) (A1)) or the caspase3 cleavage site (LQGSDEVDGSLQ (A2)) downstream of the amplified gene and a XhoI restriction site (A3) directly upstream from the amplified gene. The amplicons were inserted into the PstI and XhoI sites of the pRSET GFP2-PstI-RLuc [8], replacing native RLuc to give pRSET GFP2-RG-RLuc2, pRSET GFP2-DG-RLuc2, pRSET GFP2-RG-RLuc8, and pRSET GFP2-DG-RLuc8 where RG represents the linker sequence LQGSLVPRGSLQ and DG represents the linker sequence LQGSDEVDGSLQ. All constructs were confirmed by sequencing. The construction of GFP2-RG-RLuc was described previously [8]. All fusion proteins contain a 6 His-tag located at the N-terminus. Expression and purification of BRET-based proteins Proteins were expressed in Escherichia coli strain BL21 (DE3) (Novagen). An overnight culture was grown from a single colony in LB (10 g tryptone, 5 g yeast extract, 5 g NaCl (pH 7.4)) containing 100 lg/mL ampicillin and 2% glucose at 37 °C, 200 rpm. Expression was induced by inoculating 500 mL LB containing 100 lg/mL ampicillin to an A600 of 0.1 and incubating at 37 °C (200 rpm) for 3.5 h followed by overnight incubation at 22 °C (200 rpm). Cells were harvested 24 h after inoculation. For protein purification, cells were harvested by centrifugation at 4335g (4 °C) for 15 min and resuspended in equilibration buffer (57.7 mM Na2HPO4, 42.3 mM NaH2PO4, 300 mM NaCl, pH 7.0). The cells suspension was passed through a homogenizer (Avestin emulsiflex C3 (ATA Scientific, Australia)) at a pressure of 22,000 psi and the soluble protein fractions were isolated by centrifugation at 15,000g (4 °C) for 15 min. Proteins were purified using cobalt affinity chromatography according to the supplied instructions (BD Talon (BD Biosciences, Clontech, Australia)). Following elution of the purified protein with 150 mM imidazole, the sample was dialyzed against thrombin cleavage buffer (50 mM Tris (pH 8), 100 mM NaCl, and 1 mM EDTA) [18] or caspase-3 cleavage buffer (20 mM Pipes (pH 7.2), 100 mM NaCl, 1 mM Na-EDTA, 10 mM DTT, 0.1% (w/v) Chaps, 10% (w/v) Sucrose) [19] using a cellulose membrane (12,000 molecular weight cutoff (Sigma)). Aliquots of 500 lL protein were snap-frozen on dry ice and stored at 80 °C. Protein concentrations were determined by absorbance at 280 nm and calculated according to the method of Gill and von Hippel [20]. Instrumentation Spectral scans All spectral scans were recorded with a SpectraMax M2 plate-reading spectrofluorimeter (Molecular Devices, Australia). The reactions were carried out in 96-well plates (Perkin-Elmer,

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Australia). Bioluminescence scans of BRET2 constructs were recorded using the luminescence scan mode scanning between 360 and 650 nm. Simultaneous dual emission detection Simultaneous dual emission BRET measurements were carried out with a POLARstar OPTIMA microplate reader (BMG LabTech, Australia) using the BRET2 emission filter set comprising RLuc/ Clz400a emission filter (410 nm bandpass 80 nm) and the GFP2 emission filter (515 nm bandpass 30 nm). Thrombin assays Thrombin assays were carried out in 96-well plates by incubating purified fusion protein (1 lM) with varying amounts of thrombin protease (Quantum Scientific, Australia) in cleavage buffer (final volume of 100 lL) at 30 °C for 90 min and then recording the BRET2 signal. For BRET2 measurements 5 lM Clz400a substrate (Biosynth AG, Switzerland) was added following the 90-min period and a 0.50-s integration time was used. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS– PAGE) analysis Proteins (2.5 lg) were diluted in 1X sample loading buffer (Invitrogen, Australia) for SDS-gel electrophoresis (NuPAGE system: 12% Bis-Tris gel with Mops running buffer (Invitrogen, Australia)). After electrophoresis, the gels were washed three times in 60 ml of 45% (v/v) methanol, 10% (v/v) acetic acid for 10 min at room temperature with gentle agitation. Following the third wash, the gel was submerged in 16% (v/v) Fast Stain (Fisher Scientific, Australia) overnight with gentle shaking. Gels were destained by washing three times in 10% (v/v) acetic acid solution for 10 min with gentle shaking. Gels were photographed with transmitted fluorescent illumination using an AlphaImager 2200 video capture imaging system (Alpha Innotech Corporation). Hirudin Inhibition of thrombin protease Recombinant hirudin from yeast (Sigma, Australia) was incubated with equal units of thrombin at room temperature for 10 min prior to implementation of thrombin assays. Analysis BRET2 ratios are the ratio of integrated GFP2 channel intensity (515 nm bandpass, 30 nm) divided by the integrated RLuc/Clz400a channel intensity (410 nm bandpass, 80 nm) [7,8]. All data are reported as means ± standard deviations (SD) (n = 3) of replicate measurements made on different days on three independently produced batches of protein, unless otherwise stated. Normalized BRET2 ratios were calculated by dividing calculated BRET2 ratios following thrombin cleavage by the calculated BRET2 ratios without the addition of thrombin. Two-tailed unpaired t tests were performed using Graphpad prism (version 5.00 for Windows, Graphpad Software, San Diego, CA, USA). Statistical significance was defined as P < 0.05. Bioluminescence half-lives were estimated by nonlinear regression analysis fitting the data to a one-phase exponential decay using GraphPad Prism for Windows Version 5.00. Detection limits were calculated using [21]:

Limit of detection ðyÞ ¼ yb þ 3Sb ;

ð1Þ

where yb = a, the y-axis intercept of least squares regression line and:

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Sb ¼ Sy=x ;

ð2Þ

where

Sy=x

sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi P b 2 i ðyi  yi Þ ¼ ; n2

ð3Þ

where yi = experimental y values, ybi = fitted values to calculated least squares regression line, n = number of y values.

Limit of detection ðx valueÞ ¼ ð3  Sy=x Þ=b

ð4Þ

where b = slope of fitted least squares regression line. Results and discussion Luminescence intensity Substitution of native RLuc with either RLuc mutant increased the light output of the BRET2 fusion proteins by a factor of 10 (Fig. 1). The intensity of the integrated BRET2 signal was (3.78 ± 0.11)  103 (n = 3) relative light units (RLU)/pmol protein for GFP2-RG-RLuc, (39.13 ± 0.62)  103 RLU/pmol protein (n = 3) for GFP2-RG-RLuc2 and (36.75 ± 1.02)  103 RLU/pmol protein (n = 3) for GFP2-RG-RLuc8. This is a similar order of magnitude to the observed increase in quantum yield for the Clz400a substrate in the isolated RLuc2 and RLuc8 variants of native RLuc [11]. BRET2 ratio The BRET2 ratio, integrated GFP2 channel intensity divided by integrated RLuc/Clz400a channel intensity, of GFP2-RG-RLuc was previously calculated to be 6.21 ± 0.18 [8] compared to 3.37 ± 0.02 for GFP2-RG-RLuc2 and 3.06 ± 0.24 for GFP2-RG-RLuc8 calculated here (Fig. 2). Substitution of native RLuc with either RLuc mutant resulted in a decrease in BRET2 ratio by a factor of 2 when BRET2 components were separated by the thrombin cleavage sequence. We had anticipated that substitution of native RLuc with either RLuc mutant would increase the BRET2 ratio of the BRET2 fusion proteins based on previous reports [12] of a 5.5-fold increase in BRET2 ratio following substitution of RLuc8 for RLuc in GFP2-RGRLuc [8]. The BRET2 components in the GFP2-RLuc plasmid are separated by a multiple cloning site encoding a 16 amino acid linker (SGSSLTGTRSDIGPSR) [12] compared to a 12 amino acid linker (LQGSLVPRGSLQ) here. Our contrasting results may be due to the different natures of these linker sequences and the effect this has on relative orientations of dipoles of the BRET donor and acceptor. To further investigate the effect of the linker sequence on BRET2 energy transfer, a caspase-3 cleavage sequence was inserted

Fig.2. BRET2 ratio (mean ± SD, n = 3) of 1 lM of fusion proteins, GFP2-RG-RLuc2 and GFP2-RG-RLuc8, on addition of 5 lM Clz400a substrate following treatment (90 min, 30 °C) with 54 nM thrombin and 54 nM thrombin following pretreatment (10 min, room temperature) with 2 units of hirudin. Controls consist of 1 lM of a mixture of both donor (RLuc2 or 8) and GFP2 acceptor.

between the BRET2 donor and the acceptor to give the following fusion proteins: GFP2-DG-RLuc, GFP2-DG-RLuc2, and GFP2-DGRLuc8. Their measured BRET2 ratios were 3.77 ± 0.02 [7], 2.52 ± 0.34, and 2.39 ± 0.11, respectively. The order of the amplitudes of their BRET2 ratios, RLuc > RLuc2 > RLuc8, was the same as for the thrombin cleavage sequence. Also, all BRET2 fusion proteins incorporating the caspase-3 target sequence produced lower BRET2 ratios than the corresponding fusion proteins separated by the thrombin cleavage target sequence. We have previously attributed this to the effect of using different buffer systems to optimize cleavage conditions for each individual protease [7]. It was previously reported that replacement of native RLuc with M185V RLuc mutants improved the quantum yield, leading to BRET ratios increasing by factors of 3.4 for RLuc2 and 5.5 for RLuc8 [12]. In this study there was a decrease in BRET2 ratio when RLuc mutants were substituted for native RLuc in the BRET2 fusion proteins. This discrepancy may partly arise due to the different expression system, BRET constructs, and measurement techniques used by De et al. [12] in comparison to those used in the present study. The rate of RLuc inactivation observed in mammalian cells was an order of magnitude slower than that observed in serum [12]. In our thrombin cleavage buffer, the calculated half-life of the bioluminescence decay of GFP2-RG-RLuc was determined to be 28.9 s compared to 22.7 s for GFP2-RG-RLuc2 and 19.8 s for GFP2-RG-RLuc8 (Fig. 3). This is the same relative order (RLuc > RLuc2 > RLuc8) as for the BRET2 ratio intensities (Fig. 2) and the light intensities (Fig. 1). Thrombin cleavage

Fig.1. Emission spectra of 1 lM of the specified GFP2-RG-RLuc, GFP2-RG-RLuc2, and GFP2-RG-RLuc8 following addition of 16.7 lM Clz400a substrate (mean ± SD, n = 3). Spectral scans of multiple wells (n = 3) for each protein sample were recorded using the SpectraMax M2 spectrofluorimeter.

BRET2 assays GFP2-RG-RLuc2 and GFP2-RG-RLuc8 fusions were cleaved with 54 nM thrombin for 90 min, which changed the BRET2 ratios by factors of 18.8 ± 1.2 and 18.2 ± 0.4, respectively (Fig. 2). The final

Enhanced protease biosensor / H. Dacres et al. / Anal. Biochem. 424 (2012) 206–210

Fig.3. Decay of bioluminescence (RLU) measured using the RLuc/Clz400a emission filter (410 nm bandpass 80 nm) and an integration time of 0.5 s; bioluminescence intensity is normalized by intensity at time = 0 (mean ± SD, n = 3).

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Fig.4. Calibration of thrombin concentration with 1 lM GFP2-RG-RLuc2 and GFP2RG-RLuc8 on addition of 5 lM Clz400a substrate (mean ± SD, n = 3).

BRET2 ratios were not significantly different (P < 0.05) from those obtained by mixing 1 lM each of either donor with acceptor (control response), confirming that full cleavage had occurred (Fig. 2). Under identical assay conditions the change in BRET2 ratios is approximately the same with either of the two RLuc variants. The corresponding thrombin-catalyzed change in the BRET2 ratios of the GFP2-RG-RLuc fusion was 28.8 ± 0.20. Therefore, under identical assay conditions the change in recorded BRET2 ratios was approximately 1.5-fold higher for the original BRET2 system compared with systems incorporating RLuc2 or RLuc8. Specificity Preincubation of thrombin with two units of hirudin, which is known to stably inhibit thrombin [18], abolished any changes in BRET2 ratio for either RLuc2 or RLuc8 BRET2 systems, indicating that the change in BRET2 ratio on addition of thrombin is absolutely thrombin dependent (Fig. 2). We mixed thrombin with each purified fusion protein to characterize the cleavage products (Fig. S1, Supplementary Material). Incubation with thrombin followed by SDS–PAGE confirmed the conversion of a single 68.8-kDa band, the fusion protein, into components of 32.4 and 36.4 kDa, corresponding to His-tagged GFP2 and untagged RLuc2 (Fig. 4A) or RLuc8 (Fig. 4B). Preincubation with hirudin completely inhibited this conversion, confirming the specificity of the cleavage. Thrombin quantification The sensitivities of the two BRET2 assays were compared by incubating 1 lM of either the GFP2-RG-RLuc2 or GFP2-RG-RLuc8 fusion with increasing concentrations of thrombin (Fig. 4). This is the first report of the use of these RLuc variants in a BRET2-based in vitro assay for the quantitative determination of thrombin. The BRET2 ratio changed linearly with increasing concentrations of thrombin up to 2.7 nM for both BRET2 fusion proteins (Fig. 4). Calibrations were linear in these regions with R2 values exceeding 0.99 (Fig. 5). Comparison of the gradients of the calibrations (Fig. 5) revealed that the sensor incorporating the RLuc2 donor (y = 5.25x + 1.37) is marginally more sensitive, 1.2-fold, to changing thrombin concentrations than the one with RLuc8 (y = 4.49x + 1.33). The calculated detection limits (see Materials and methods (Eqs. (1)–(4)) for formulas and explanation) for thrombin were 0.23 and 0.26 nM for RLuc2 and RLuc8 BRET2 systems, respectively. Both BRET2 systems

Fig.5. Linear calibration of thrombin concentration with 1 lM GFP2-RG-RLuc2 and GFP2-RG-RLuc8 on addition of 5 lM Clz400a substrate (mean ± SD, n = 3).

described here are an order of magnitude less sensitive for thrombin detection than the native RLuc BRET2 system, which had a detection limit of 0.015 nM and a dynamic range extending up to 5.4 nM [7] but an order of magnitude more sensitive than the FRET system [8] (Table S2, Supplementary Material). The detection limit for BRET detection of MMP-7 cleavage of RLuc8-QD655 linked by a MMP specific substrate was 0.26 nM [13], a similar sensitivity to the mutant BRET2 systems characterized here. Although BRET2 systems incorporating RLuc2 and 8 have lower sensitivities and higher detection limits for thrombin than the native RLuc BRET2 system they are both sufficiently sensitive for measuring thrombin concentrations associated with plasma clots, estimated to be 0.46 nM [22]. Identifying acceptor molecules with large Stokes shifts that maintain a good spectral overlap with the RLuc donor emission spectrum may offer the greatest improvement to the sensitivity of BRET2-based assays. For instance, a novel BRET system (BRET3), combining a mutant red fluorescent protein (mOrange) with RLuc8 and the BRET2 substrate donor exhibited several-fold improvement over existing BRET fusion proteins, including GFP2-RLuc8 [23]. An alternative approach would be to use novel Clz400a derivatives, which have been shown to improve the signal to noise ratio and the emission lifetime of the GFP-RLuc8 BRET2 system while maintaining good spectral resolution between donor and acceptor emission peaks [24].

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Conclusions We conclude that the increase in luminescence afforded to the BRET2 system by substitution of native RLuc by RLuc2 or RLuc8 in practice comes at the expense of assay sensitivity. However, overall these mutant systems are still more sensitive than FRET and brighter than standard BRET2. The increase in luminescence with the modified BRET2 systems could lead to shorter acquisition times and allows the use of lower amounts of protein and lower substrate concentrations, which would have considerable practical advantages. This study highlights the fact that it is inadvisable to draw conclusions based solely on the performance of individual BRET components without testing how the system as a whole performs. Acknowledgments The authors gratefully acknowledge Dr. Colin Scott and Dr. Peter Campbell for advice and critical review of the manuscript. We are grateful to S. Gambhir (Stanford University, CA, USA) and K.D.G. Pfleger (Western Australian Institute of Medical Research and Centre for Medical Research, University of Western Australia, Perth, Australia) for providing cDNA constructs.

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