Bacterial Expression and Re-Engineering of Gaussia Princeps Luciferase and Its Use As a Reporter Protein

Biochimica et Biophysica Acta 1854 (2015) 1392–1399

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Review Bacterial expression and re-engineering of Gaussia princeps luciferase and its use as a reporter protein

Nan Wu, Tharangani Rathnayaka, Yutaka Kuroda ⁎

Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei-shi, Tokyo 184-8588, Japan article info abstract

Article history: Bioluminescence, the generation of visible light in a living organism, is widely observed in nature, and a large va- Received 31 March 2015 riety of bioluminescent proteins have been discovered and characterized. Luciferase is a generic term for biolu- Received in revised form 24 April 2015 minescent enzymes that catalyze the emission of light through the oxidization of a luciferin (also a generic Accepted 15 May 2015 term). Luciferase are not necessarily evolutionary related and do not share sequence or structural similarities. Available online 27 May 2015 Some luciferases, such as those from ﬁreﬂies and Renilla, have been thoroughly characterized and are being

Keywords: used in a wide range of applications in bio-imaging. Gaussia luciferase (GLuc) from the marine copepod Gaussia Bioluminescence imaging princeps is the smallest known luciferase, and it is attracting much attention as a potential reporter protein. GLuc Protein folding identification is relatively recent, and its structure and its biophysical properties remain to be fully characterized. Protein engineering Here, we review the bacterial production of natively folded GLuc with special emphasis on its disulfide bond for- Protein domain mation and the re-engineering of its bioluminescence properties. We also compare the bioluminescent proper- Disulfide bonds ties under a strictly controlled in vitro condition of selected GLuc's variants using extensively purified proteins Bioinformatics with native disulfide bonds. Furthermore, we discuss and predict the domain structure and location of the cata- Structure prediction lytic core based on literature and on bioinformatics analysis. Finally, we review some examples of GLuc's emerg- ing use in biomolecular imaging and biochemical assay systems. © 2015 Elsevier B.V. All rights reserved.

1. Introduction essential bioluminescence marker for in vivo imaging [8–10]. A physi- cally and chemically stabilized RLuc variant with improved biolumines- Luciferase is a generic name for enzymes that emit light by catalyz- cence properties [11] and its X-ray crystal structure was reported [12]. ing substrates referred to as luciferin ([1], pages xix–xxi). A large variety In addition to the well known FLuc and RLuc, luciferases from various of luciferases from different biological and evolutionary origins have organisms having distinct sequences, structures, and biochemical prop- been identified, however, few have been thoroughly characterized and erties have also been reported [13–16] (Table 1). fewer still have been cloned and used in analytical applications. The Gaussia luciferase (GLuc) is a coelenterazine-dependent luciferase most commonly used luciferase in experimental imaging applications (Table 1)identified from the marine copepod Gaussia princeps [15] originates from the North American firefly(Photinus pyralis) [2].Firefly (Fig. 1), which lives in temperate and tropical waters worldwide [17]. luciferase (FLuc)'s molecular weight is 62 kDa (Table 1) [3].Itcatalyzes GLuc, which catalyzes the production of a bright blue light, is secreted the substrate benzothiazole (D-luciferin) in the presence of oxygen, ATP by Gaussia princeps in combination with rapid swimming probably as and Mg+2, and emits green light with a peak emission of 562 nm [4].Its a defense mechanism [18]. GLuc's sequence (mRNA, complete cds) structure has been solved by X-ray crystallography [5].SeveralFLucvar- was first reported in 1999 [15]. It is presently attracting much attention iants with different luminescence characteristics have been designed, for bio-imaging applications because of its small size and strong lumi- which has enlarged its application range [6]. nescence activity, as demonstrated by over two hundred recently re- Renilla luciferase (RLuc) derived from sea pansy (Renilla reniformis) ported examples. Here, we review literature on GLuc, with a special is also well characterized. It has a molecular weight of 36 kDa [3,7], emphasis on the formation of disulfide bonds in bacterially expressed and it catalyzes coelenterazine to produce the excited coelenteramide GLuc and its functional re-designing. In addition, we discuss its structur- state (Table 1) emitting a blue light with a peak emission of 480 nm. Un- al and functional properties in the light of the literature and of bioinfor- like FLuc, RLuc does not require ATP for activity, and it is becoming an matics analysis of its amino acid sequence.

2. Biochemical properties of GLuc Abbreviations: ATP, adenosine triphosphate; HPLC, high performance liquid chromatography ⁎ Corresponding author. Tel./fax: +81 42 388 7794. With 185 amino acids and a molecular mass of 19.9 kDa, GLuc is E-mail address: [email protected] (Y. Kuroda). the smallest luciferase reported so far (Table 1, Fig. 4A). GLuc's

Table 1 Comparison of GLuc with selected luciferases.

Species Size Molecular Wt Cofactors Substrate Isoelectric Cysteine number Reference (aa) (kDa) point (frequency, %)

Gaussia princeps (Copepod) 185 20 None Coelenterazine 6.8 11 (5.9) [3] +2 Photynus pyralis (Firefly) 550 62 ATP, Mg D-Luciferin (benzothiazole) 6.6 4 (0.7) [3] Renilla reniformis (Sea pansy) 311 36 None Coelenterazine 5.8 3 (1.0) [3] Oplophorus 196 21 None Coelenterazine 5.0 1 (0.5) [16] Pleuromamma 198 22 N/A N/A 6.6 17 (8.6) [15] Vibrio harveyi (LuxA) 355 37 Long-chain aldehyde Reduced riboflavin phosphate (FMNH2) 4.8 8 (2.2) [13,64] Lingulodinium polyedrum 1241 137 None Dinoflagellate luciferin 7.3 29 (2.3) [14]

FLuc contains four free cysteines. Little influence on its luminescence was observed by changing them to serine [65]. RLuc contains three free cysteines, and two of them affect its luminescence property when mutated to alanine [66]. bioluminescence intensity is significantly stronger (over 200 fold) than Verhaegen M and Christopoulos T.K. [18].TheydesignedanE. coli that of FLuc and RLuc [3], which are commonly used as reporter pro- over-expression system that produces a biotinylated bap (biotin accep- teins. Alike RLuc, GLuc catalyzes the substrate, coelenterazine, to pro- tor peptide)-GLuc fusion protein. The final yield of GLuc purified by Av- duce the excited state of coelenteramide by emitting blue light (Fig. 2) idin affinity chromatography from the soluble fraction of the E. coli with a peak emission at 480 nm [3]. Unlike FLuc, GLuc does not require culture was 0.55–0.69 mg/L, but nearly 60% of the luminescence was ATP or other co-factors for activity. The bioluminescence activity of lost during affinity chromatography purification [18]. GLuc is highly stable under a wide range of temperatures (90% and 65% of the luminescence intensity is retained after a 30-minute incuba- 4. Multiple disulfide bonds and expression of native GLuc in E. coli tion at 60 °C and 90 °C, respectively) [19,20] and in an acidic environ- ment (down to pH 1.5) [21]. GLuc's bioluminescence is salt GLuc contains eleven cysteines corresponding to an amino acid fre- concentration dependent [19,22] and activity reaches a maximum at quency of 5.95%, which is in sharp contrast to FLuc and RLuc, containing 50 mM NaCl [19]. GLuc's bioluminescence is strongly inhibited by respectively four and three non-disulfide bonded cysteines (Table 1). heavy metal ions (Cu2+) and stimulated by monovalent ions (Cl−,I−, Ten of the GLuc's cysteines form disulfide bonds [22,28], whereas the Br−) [22]. GLuc is highly specific for coelenterazine, and shows narrow 11th cysteine is located in the pro-sequence, which presumably helps substrate specificity unlike RLuc, which catalyzes coelenterazine, and GLuc's folding by reshuffling non-native disulfide bonds in vivo [29, also several analogs as well [23]. 30]. Disulfide bonded proteins are usually difficult to express in E. coli in a native form, because E. coli 's cytoplasm provides a reducing envi- ronment, and cysteines are air-oxidized upon cell breakage and during 3. Expression of recombinant GLuc purification, often resulting into non-native disulfide bonds [27].Even if the cysteines were oxidized in the cytoplasm, E. coli does not have Various expression systems for GLuc have been reported. GLuc was the molecular machinery to reshuffle non-native disulfide bonds into cloned and expressed in mammalian [3,24], bacterial [21], insect [25], the correct ones, and most multi-disulfide proteins thus turn into func- and green algae [26] cells. Expression in Escherichia coli (E. coli)isanim- tionally inactive aggregates [27]. portant step toward re-designing a protein, not only because it provides To overcome the problem of non-native disulfide bonds, GLuc was a handy and inexpensive method for producing recombinant proteins, expressed in the E. coli periplasm using a prokaryotic secretion signal but also and foremost because genetic modifications are most easily in- peptide, the pelB leader sequence [31,32]. However, the yield and spe- troduced and assessed with this system [27]. To our knowledge, the first cific activity of the secreted GLuc were relatively low (250 μg/200 mL quantitative analysis of GLuc expression in E. coli was reported by culture), and the pelB cleavage during protein expression in E. coli was inefficient [31]. Cell free systems, where the protein folding environ- ment can be controlled, have been used in an attempt to assist native disulfide bonds formation [28,33]. In principle, some proteins can be recovered from insoluble aggregates by optimizing the refolding conditions [34]. However, with as much as five disulfide bonds, the refolding of GLuc appears to be a difficult task [19]. Finally, low expression tem- perature (15 °C) increases the fraction of natively folded GLuc, but also decreases the final protein yield [19]. Formation of non-native disulfide bonds in recombinant proteins is closely related to the low solubility of miss-folded proteins. Aggregation often competes with the folding reaction [20], but GLuc, like most small proteins, is intrinsically able to fold into a native structure [29]. Thus, a GLuc with enhanced solubility is anticipated to increase the yield of well folded protein with native-like disulfide bonds. Recently, various attempts for increasing the solubility of GLuc have been reported. In one of them, GLuc was fused to a highly soluble IgG-binding domain of protein A (ZZ-domain, 116 amino acid residues) [35], but it remains unclear whether the fused GLuc was solubilized in a native, fully functional form with the correct disulfide bonds. On the other hand, we constructed a GLuc variant with a Solubility Enhancement Peptide tag (SEP tag [36–38]), consisting of nine aspartic acid tags, attached to its C terminus, which can significantly increase protein solubility. As a result, Fig. 1. Picture of Gaussia princeps by Professor Russel Hopcroft, University of Alaska — Fair- 98% of the tagged GLuc was expressed in the soluble fraction at 25 °C, banks (UAF), and reproduced with the author's permission. which was fully functional and formed native-like disulfide bonds [20]. 1394 N. Wu et al. / Biochimica et Biophysica Acta 1854 (2015) 1392–1399

Fig. 2. Luciferin — Luciferase reaction catalyzed in Gaussia princeps,O2 binds to the 2-position of the coelenterazine molecule, which is peroxided to form a 4-membered ring “dioxeranone”, which is decomposed to produce CO2, and produces an amide anion in the excited state of coelenteramide. This excited coelenteramide releases its energy by emitting blue light [1] pp: 168–172.

5. Mutational analysis of GLuc activity mutations which prolonged the luminescence time are most probably located on the GLuc's molecular surface as demonstrated by a click at- The luminescence of the wild type GLuc (wt-GLuc) decays within tachment reaction [33]. seconds, which can potentially limit its use as a reporter protein. Re- Detergents may interfere with the imaging or screening processes by cently, several GLuc variants with prolonged bioluminescence have denaturing the protein or affecting its interactions with the substrate. thus been designed [31,33] (Fig. 3). For example, a single mutation at Thus, in many applications, mutants exhibiting prolonged luminescence residue 43 extended the light emission half life from 2.4 min for the in a detergent-free condition are required. GLuc4, which has 3 muta- wt-GLuc to 9.1 min for the GLuc-M43I mutant, when assayed in vitro tions (L30P, L40P, M43V), exhibits the longest light emission half-life in the presence of Triton X-100 [31] (Fig. 3;mutationA).However, (Fig. 3; mutation C). Its bioluminescence intensity is 1/100 that of wt- the specific activity of this mutation was three fold lower than that of GLuc but it does not require detergent for prolonged bioluminescence, the wild type GLuc. Welsh J.P. et al. reported that the combination of which is essential for in-vivo bio-imaging [39]. two mutations (M43L and M110L) extended the light emission half Recently, Kim S.B. et al. constructed a GLuc variant with strong life to 14 min without reduction of the specific activity, when measured and prolonged bioluminescence by extensively mutating 16 sites in the presence of a detergent (Tween-20) (Fig. 3; mutation B). These chosen based on experimental results and semi-rational consider- ations [40]. They found that F72, I73, H78, Y80, and I91, significantly alter the bioluminescence intensity. Further, they reported that the peak emission of the I73L and MONSTA (F72W/I73L/H78E/Y80W) (Fig. 3; mutation D) variants were shifted by, respectively, 16 nm and 33 nm, making GLuc more attractive for bio-imaging applications [41].

6. GLuc's two domains and their functional interplay

Though options for rationally redesigning GLuc are presently limited due to the lack of structural data, mutational analysis is starting to reveal its structural and functional characters. Inouye and co-workers have ex- perimentally identified two putative domains (27–97, 98–168) in GLuc (Fig. 4A), which exhibit residual bioluminescence activity in isolation [22]. So far, except for residues 110, all of the reported mutations that im- pact bioluminescence are located in the hydrophilic region of domain 1 Fig. 3. Hydropathy plot of GLuc. The Kyte–Doolittle hydropathy scale [67] are averaged (Fig. 3). Domain 2 also possesses hydrophilic regions, which are candi- using a 21 residue window, therefore, values for the first and last 10 amino acids are not date locations for the catalytic core [22]. Further, cooperative binding available. Residue numbering excludes the secretion tag. The location of the putative do- of luciferase to GLuc is suggested by a Hill coefficient of 2.9, which con- mains is shown by arrows on the top. “A”,“B”,“C”,and“D” indicate mutations that signifi- trasts with a Hill coefficient of 1.1 for RLuc reflecting a one-to-one non- cantly changed GLuc's bioluminescence. “A” indicates the M43I mutation “B” indicates both the M43L and M110L mutations. “C” indicates the L30P, L40P, and M43V. “D” indi- cooperative binding [42]. A synergetic extension of light emission life cates the MONSTA's mutations (F72W, I73L, H78E, Y80W). time observed by simultaneously mutating M43 and M110 to L (Fig. 3; N. Wu et al. / Biochimica et Biophysica Acta 1854 (2015) 1392–1399 1395

Fig. 4. (A) Sequence alignment, secondary structure prediction and domain information of GLuc. GLuc indicates the GLuc's amino acid sequence. Residues −16 to 0 represent GLuc's secretion tag sequence. Clustal Co indicates amino acid conservation throughout the 12 sequences listed in Fig. 4B according to Clustal W: (An asterisk indicates a fully conserved residue, a column shows a highly conserved residue, and a dot shows a poorly conserved residue according to Clustal W classiﬁcation). Domain 1 shows GLuc's domain 1 sequence alignment against that of domain 2. Domain Co indicates amino acid conservation throughout the 12 sequences and domain 1 and domain 2. SS 85% shows GLuc secondary structure predicted by 7 predictors with 85% consensus (Supplemental data SFig 1). (B) Phylogenetic tree topology of GLuc and selected 12 similar luciferases constructed by maximum likelihood using MEGA6 [68].GLucismarkedwithanopen diamond. The origin, residue number, and sequence identity are indicated. Branch length represents the number of residue substitutions per site. mutation B), which are counterpart residues in domains 1 and 2 [33], the second domain corresponding to those mutated in MONSTA (F72/ further suggests that the catalytic core of GLuc is located at the interface I73/H78/Y80 [41])(Fig. 3; mutation D) might further red-shift the emis- of the two domains [42]. Thus we speculate that mutating residues in sion spectrum of GLuc. 1396 N. Wu et al. / Biochimica et Biophysica Acta 1854 (2015) 1392–1399

predicted, using the same server, as a single domain protein, consistent with its crystal structure (Protein Data Bank ID 2PSE). As a further bioinformatics support for the two-domain hypothesis, a domain linker [44, 45] is predicted at residues 98–109 (Fig. 5) both with DLP-SVM [46] and DROP [47]. Multiple sequence analysis also suggested the presence of two conserved sequence regions in GLuc. First, a Basic Local Alignment Search Tool (BLAST) [48] search against the Uniprot KB/Swiss-Prot dataset identified 12 luciferases with significant sequence identities and E-values below 10−30 (Fig. 4B). Further, multiple sequence alignment using ClustalW [49] indicates two highly conserved regions corresponding to residues 32–80 and 99–158, corroborating the presence of two domains reported by Inouye et al. [22]. We further aligned GLuc's putative domain 1 against its own putative domain 2 as well as the above 12 luciferases expecting to narrow the conserved region. The calculation indicated that residues 52–77 and 123–148 were the most conserved regions, suggesting that GLuc's active site might be located there (Fig. 4A). The above discussion suggests that 5 cysteines are distributed in each putative domain, and one in the secretion tag. Except for the one Fig. 5. Schematics of GLuc molecular and structural information. A pre-sequence extending from residues 1 to 26 is predicted to precede two putative structural domains (Domain in the secretion tag, the cysteines are highly conserved strongly suggest- 1: 27–97; Domain 2: 98–168). Overall, GLuc is predicted to be a helical protein, and se- ing that the 5 disulfide bonds are important for forming the native GLuc quence alignment predicts that each domain contains two intra-domain disulfide bonds structure. Sequence alignment of domain 1 against 2 indicates a strong (shown by broken lines) and an inter-domain disulfide bond between cysteines C59 and correspondence between C52, C56, C65, and C77 in domain 1 and C123, C120. Further, a domain linker extending from residues 98 to 109 was predicted using C127, C136, and C148 located in domain 2. This suggests first that the DLP-SVM and DROP, which overlaps with the start of the domain 2 region (98–168). Re- gions 52–77 in domain 1 and 123–148 in domain 2 are highly conserved (shown by a two domains possess a similar fold and second that the two remaining gray ellipse) and are anticipated to overlap with the active site. The activity is regulated cysteines (C59 in domain 1 and C120 in domain 2) form an inter- by residues located in both domains in a synergetic manner suggesting that the active domain disulfide bond (Fig. 4A, 5). site lies between the two putative domains. 8. Assessment of yield and bioluminescence of purified GLuc variants

7. Bioinformatics characterization of GLuc's sequence and structure Here we compare the properties of some selected GLuc variants that we and others developed in previous studies. We examined their expres- In the absence of experimental structural information, bioinformat- sion levels and purification yields, and measured their bioluminescence ics prediction of GLuc's structural properties provide unique clues for under a well controlled in vitro condition and constant protein concentra- designing variants with improved functional or biochemical properties. tion (Table 2). All variants were expressed in E. coli BL21 (DE3) and puri- This section summarizes some of GLuc's structural properties predicted fied using a nickel resin column. The proteins were further purified using using standard bioinformatics tools. First, the consensus secondary reversed phase high-performance liquid chromatography (HPLC) to re- structures of GLuc predicted using seven predictors indicate that GLuc move miss-folded GLucs forming miss-matched disulfide bonds. The con- is an α-helical protein with 30% helices, 4% sheets, 36% coils, and 29% centration of natively folded GLucs was measured by Bradford protocol without consensus prediction (the coil and no prediction group totals before luminescence activity measurement [19]. 60%; Supplemental data SFig1), which is in line with our previously re- First, we examined mutations that improved the expression levels of ported secondary structure content calculated from circular dichroism's native GLuc in E. coli. The purification yield of GLuc was improved by ap- measurement (30% helices, 12% sheets and 58% coils) [19] (Fig. 4A). proximately 40% (as compared to GLuc-C0D; see Table 2) by the addi- In addition, GLuc is predicted to form two domains according to the tion of a hydrophilic tail [36] containing nine aspartic acids [38] to its TRUST [43] domain prediction server, which may reflect Inouye et al.'s ex- C terminus (GLuc-C9D). This result underscores the hypothesis that perimental finding of two functional domains. As a control, RLuc is the increase of GLuc's solubility provides time for its cysteines to form

Table 2 Protein yield and in vitro activity of four selected GLuc variants.

Variant Vector Detailed description Characteristics Total Emission Intensity at Relative yield peak emission peak activity [mg/200 (nm) [%]b mL]a

GLuc-C0D pAED4 His-GLuc Wild type GLuc with histidine tag (H6) 0.20 480 219 98

GLuc-C9D pAED4 His-GLuc-C9D A C9D-tag (G(GD3)3) is attached at the C termini in 0.28 480 224 100 order to increase protein solubility [20] GLuc-TG pET21c His-FactorXa-TG-GLuc variant FactorXa sites were inserted for tags cleavage. 0.73 480 224 100 (E100A/G103R)-FactorXa-TMG-C9D Mutations E100A and G103R were inserted for increasing protein expression level. TG and TMG were inserted for improving cleavage efﬁciency. GLuc-MONSTA pET21c His-FactorXa-TG-GLuc F72W/I73L/H78E/Y80W increase the intensity and 1.0 490 250 112 (E100A/G103R/F72W/I73L/H78E/Y80W)- red shift the emission spectrum by about 30 nm FactorXa-TMG-C9D according to [41]

Relative activity of the native peak collected from HPLC and determined at 20 °C by mixing 30 μLofcoelenterazineatafinal concentration of 3 μMtoa2mL0.01μMGLucsolutionin50mM Tris–HCl buffer pH 8.0 and 50 mM NaCl. a Protein yield calculated from the surface area under the native peak of reverse phase HPLC. b Normalized activity using the protein concentration of GLuc-C9D determined in (a)asareference. N. Wu et al. / Biochimica et Biophysica Acta 1854 (2015) 1392–1399 1397 the native disulfide bonds. Next, some modifications of the expression introducing the mutations reported by Kim et al. (F72W/I73L/H78E/ vector increased protein expression, as demonstrated in GLuc-TG's ex- Y80W; Table 2). MONSTA reportedly emits a blue-greenish light, pression which was about three times higher than that of GLuc-C9D which has a 33 nm red-shifted peak emission, and it exhibits a ten- (Table 2). The bioluminescence activities of all the above three variants fold increased luminescence intensity, according to Kim et al. However, were identical to that of GLuc-C0D (Table 2, Fig. 6A). our measurements carried out under a strictly controlled in vitro condi- Next, we examined mutations that modified the bioluminescence tion with HPLC-purified proteins indicated only a 10 nm red-shift. We properties of GLuc. We thus re-constructed GLuc-MONSTA by also found that the luminescence intensity of GLuc-MONSTA as measured at the peak emission was virtually identical to that of the wild type GLuc (Fig. 6B). This discrepancy might arise from the measurement conditions, which were carried out in the cell slurry in Kim's experiment and also from the putative mixture of GLuc with miss-matched disulfide bonds in Kim's experiment [41]. Nevertheless, MONSTA remains so far one of the most successfully engineered GLuc variants. Besides the peak emission wavelength, and more generally the emission spectra, the intensity and time dependence of the emitted light are essential properties for characterizing the bioluminescence of a reporter protein. In our measurement, GLuc-MONSTA's bioluminescence intensity decreased faster than that of GLuc (Fig. 6C). After 50 s, GLuc-MONSTA retained only 5% activity, which contrasted with the 17% activity retained by wild type GLuc.

9. Bio-imaging applications of GLuc

Imaging using bioluminescent proteins is a very sensitive, non- invasive, and non-toxic method that can be adapted to high- throughput studies [50]. Here we briefly mention some of them in order to illustrate the potentials of this enzyme as a reporter protein. Examples of applications, include the real-time tracking of tumor cells and cell metastasis [3,32,51–54], the monitoring of fungal infections (such as Candida albicans and Aspergillus fumigates [55,56]) in living ani- mals, or the study of viral behavior within cells [57,58]. Further, as a nat- urally secreted enzyme, GLuc is ideal for monitoring biological processes directly in conditioned media (blood, serum or urine) [32,59].GLuchas also been used to monitor intracellular Ca2+ levels [40], and for the detection of protein–protein interaction both in vivo and in vitro [60]. Recently, GLuc is used in combination with other luciferases (firefly, click beetle, and Renilla luciferases), to develop double- [10,61] and triple-reporters [62] for simultaneously probing multiple protein interactions within the same cell. Finally, GLuc has been used as the donor for bioluminescence resonance energy transfer (BRET) and enhanced yel- low fluorescence protein (EYFP) was used as the acceptor to detect pro- teolysis in cells [63].

10. Conclusion

GLuc, from the marine copepod Gaussia princeps, is the smallest known luciferase and its identification is relatively new. Recently, we established a GLuc expression system in E. coli by optimizing the formation of native disulfide bonds, which is expected to accelerate the design of GLuc variants and thus widen its range of applications. Biophysical characterization of purified GLuc indicated a highly stable protein that remains active even when exposed to high temperatures. Though the high resolution tertiary structure remains to be determined, secondary structure prediction, corroborated by circular dichroism experiments, Fig. 6. Bioluminescence spectra of GLuc variants. (A) Emission spectra of GLuc-C0D, GLuc- predicts a helical protein. Mutational and bioinformatics analyses C9D and GLuc-TG. GLuc-C0D, GLuc-C9D and GLuc-TG are shown by a solid line, broken line, and dotted line, respectively. Bioluminescence was initiated by adding 30 μLof strongly suggest the presence of two structural domains, each contain- coelenterazine to a final concentration of 3 μMtoa2mL0.01μM GLuc solution in 50 mM ing highly conserved 20 to 25 residues long sequences that are possibly Tris–HCl buffer pH 8.0 and 50 mM NaCl. The spectra were measured at 20 °C using a Jasco critical for bioluminescence activity. FP-6500 fluorescence spectrophotometer with a scan speed of 5000 nm/min and an emission bandwidth of 1 nm. (B) Emission spectrum of GLuc-TG and GLuc-MONSTA (relative intensity %). GLuc-TG and GLuc-MONSTA are shown by a dotted line and solid line, Acknowledgements respectively. The spectra were measured under the same conditions as Fig. 6A but with a scan speed of 20,000 nm/min and an emission bandwidth of 5 nm. (C) Time dependence We thank members of the Kuroda Laboratory for discussion and help of bioluminescence activity. GLuc-TG and GLuc-MONSTA are shown by a dotted line and with bioinformatics analysis and protein characterization. We are grateful solid line, respectively. The experimental procedures and sample conditions are the same to Professor Russel Hopcroft, University of Alaska–Fairbanks (UAF), for as those used in Fig. 6A. The intensities of the luminescence were measured every second, where time zero corresponds to the addition of coelenterazine, at the respective peak emis- kind permission to reproduce his photograph of Gaussia princeps (Fig. 1; sion (GLuc-TG: 480 nm; GLuc-MONSTA: 490 nm). R. Hopcroft copyright), and Patricia McGahan for English proofreading. 1398 N. Wu et al. / Biochimica et Biophysica Acta 1854 (2015) 1392–1399

This work was supported by a JSPS grant-in-aid for scientific research [26] O. Ruecker, K. Zillner, R. Groebner-Ferreira, M. Heitzer, Gaussia-luciferase as a sensitive reporter gene for monitoring promoter activity in the nucleus of the green alga (KAKENHI: 23651213, 26560432). Nan Wu was supported by an honor's Chlamydomonas reinhardtii, Mol. Genet. Genomics 280 (2008) 153–162. scholarship from the Japan Student Services Organization (JASSO). [27] F. Baneyx, M. Mujacic, Recombinant protein folding and misfolding in Escherichia coli, Nat. Biotechnol. 22 (2004) 1399–1408. [28] A.R. Goerke, A.M. Loening, S.S. Gambhir, J.R. Swartz, Cell-free metabolic engineering Appendix A. Supplementary data promotes high-level production of bioactive Gaussia princeps luciferase, Metab. Eng. 10 (2008) 187–200. [29] C.B. Anfinsen, Principles that govern the folding of protein chains, Science 181 Supplementary data to this article can be found online at http://dx. (1973) 223–230. doi.org/10.1016/j.bbapap.2015.05.008. [30] J.S. Weissman, P.S. Kim, The pro region of BPTI facilitates folding, Cell 71 (1992) 841–851. [31] C.A. Maguire, N.C. Deliolanis, L. Pike, J.M. Niers, L.A. Tjon-Kon-Fat, M. Sena-Esteves, References B.A. Tannous, Gaussia luciferase variant for high-throughput functional screening applications, Anal. Chem. 81 (2009) 7102–7106. [1] O. Shimomura, Bioluminescence: Chemical Principles and Methods, World Scientif- [32] B.A. Tannous, Gaussia luciferase reporter assay for monitoring biological processes ic, 2006. (Place Published). in culture and in vivo, Nat. Protoc. 4 (2009) 582–591. [2] J.R. de Wet, K.V. Wood, D.R. Helinski, M. DeLuca, Cloning of fireflyluciferasecDNA [33] J.P. Welsh, K.G. Patel, K. Manthiram, J.R. Swartz, Multiply mutated Gaussia lucifer- and the expression of active luciferase in Escherichia coli, Proc. Natl. Acad. Sci. U. S. ases provide prolonged and intense bioluminescence, Biochem. Biophys. Res. A. 82 (1985) 7870–7873. Commun. 389 (2009) 563–568. [3] B.A. Tannous, D.E. Kim, J.L. Fernandez, R. Weissleder, X.O. Breakefield, Codon- [34] A. Kato, M. Yamada, S. Nakamura, S. Kidokoro, Y. Kuroda, Thermodynamic proper- optimized Gaussia luciferase cDNA for mammalian gene expression in culture and ties of BPTI variants with highly simplified amino acid sequences, J. Mol. Biol. 372 in vivo, Mol. Ther. 11 (2005) 435–443. (2007) 737–746. [4] W.D. McElroy, The energy source for bioluminescence in an isolated system, Proc. [35] S. Inouye, Y. Sahara, Soluble protein expression in E. coli cells using IgG-binding do- Natl. Acad. Sci. U. S. A. 33 (1947) 342–345. main of protein A as a solubilizing partner in the cold induced system, Biochem. [5] E. Conti, N.P. Franks, P. Brick, Crystal structure of firefly luciferase throws light on a Biophys. Res. Commun. 376 (2008) 448–453. superfamily of adenylate-forming enzymes, Structure 4 (1996) 287–298. [36] A. Kato, K. Maki, T. Ebina, K. Kuwajima, K. Soda, Y. Kuroda, Mutational analysis of pro- [6] B.R. Branchini, T.L. Southworth, N.F. Khattak, E. Michelini, A. Roda, Red- and green- tein solubility enhancement using short peptide tags, Biopolymers 85 (2007) 12–18. emitting firefly luciferase mutants for bioluminescent reporter applications, Anal. [37] M.M. Islam, M.A. Khan, Y. Kuroda, Analysis of amino acid contributions to protein Biochem. 345 (2005) 140–148. solubility using short peptide tags fused to a simplified BPTI variant, Biochim. [7] W.W. Lorenz, R.O. McCann, M. Longiaru, M.J. Cormier, Isolation and expression of a Biophys. Acta 1824 (2012) 1144–1150. cDNA encoding Renilla reniformis luciferase, Proc. Natl. Acad. Sci. U. S. A. 88 (1991) [38] M.A. Khan, M.M. Islam, Y. Kuroda, Analysis of protein aggregation kinetics using 4438–4442. short amino acid peptide tags, Biochim. Biophys. Acta 1834 (2013) 2107–2115. [8] A.M. Loening, A. Dragulescu-Andrasi, S.S. Gambhir, A red-shifted Renilla luciferase [39] M.H. Degeling, M.S. Bovenberg, G.K. Lewandrowski, M.C. de Gooijer, C.L. Vleggeert- for transient reporter-gene expression, Nat. Methods 7 (2010) 5–6. Lankamp, M. Tannous, C.A. Maguire, B.A. Tannous, Directed molecular evolution re- [9] O. Gheysens, I.Y. Chen, M. Rodriguez-Porcel, C. Chan, J. Rasooly, C. Vaerenberg, R. veals Gaussia luciferase variants with enhanced light output stability, Anal. Chem. Paulmurugan, J.K. Willmann, C. Deroose, J. Wu, S.S. Gambhir, Non-invasive biolumi- 85 (2013) 3006–3012. nescence imaging of myoblast-mediated hypoxia-inducible factor-1 alpha gene [40] S.B. Kim, M. Sato, H. Tao, Split Gaussia luciferase-based bioluminescence template transfer, Mol. Imaging Biol. 13 (2011) 1124–1132. for tracing protein dynamics in living cells, Anal. Chem. 81 (2009) 67–74. [10] R. Huang, J. Vider, I. Serganova, R.G. Blasberg, ATP-binding cassette transporters [41] S.B. Kim, H. Suzuki, M. Sato, H. Tao, Superluminescent variants of marine luciferases modulate both coelenterazine- and D-luciferin-based bioluminescence imaging, for bioassays, Anal. Chem. 83 (2011) 8732–8740. Mol. Imaging 10 (2011) 215–226. [42] G. Tzertzinis, E. Schildkraut, I. Schildkraut, Substrate cooperativity in marine lucifer- [11] A.M. Loening, A.M. Wu, S.S. Gambhir, Red-shifted Renilla reniformis luciferase vari- ases, PLoS One 7 (2012) e40099. ants for imaging in living subjects, Nat. Methods 4 (2007) 641–643. [43] R. Szklarczyk, J. Heringa, Tracking repeats using significance and transitivity, Bioin- [12] A.M. Loening, T.D. Fenn, S.S. Gambhir, Crystal structures of the luciferase and green formatics 20 (Suppl. 1) (2004) i311–i317. fluorescent protein from Renilla reniformis, J. Mol. Biol. 374 (2007) 1017–1028. [44] S. Miyazaki, Y. Kuroda, S. Yokoyama, Identification of putative domain linkers by a [13] R. Belas, A. Mileham, D. Cohn, M. Hilman, M. Simon, M. Silverman, Bacterial biolumi- neural network — application to a large sequence database, BMC Bioinformatics 7 nescence: isolation and expression of the luciferase genes from Vibrio harveyi,Sci- (2006) 323. ence 218 (1982) 791–793. [45] T. Tanaka, S. Yokoyama, Y. Kuroda, Improvement of domain linker prediction by in- [14] L. Li, J.W. Hastings, The structure and organization of the luciferase gene in the pho- corporating loop-length-dependent characteristics, Biopolymers 84 (2006) tosynthetic dinoflagellate Gonyaulax polyedra, Plant Mol. Biol. 36 (1998) 275–284. 161–168. [15] B.J.B. C.S., Sent-Gyorgyi, Luciferases, Fluorescent Proteins, Nucleic Acids Encoding [46] T. Ebina, H. Toh, Y. Kuroda, Loop-length-dependent SVM prediction of domain the Luciferases and Fluorescent Proteins and the Use Thereof in Diagnostics, High linkers for high-throughput structural proteomics, Biopolymers 92 (2009) 1–8. Throughput Screening and Novelty Items, International Pub., 1999 [47] T. Ebina, H. Toh, Y. Kuroda, DROP: an SVM domain linker predictor trained with op- [16] S. Inouye, K. Watanabe, H. Nakamura, O. Shimomura, Secretional luciferase of the lu- timal features selected by random forest, Bioinformatics 27 (2011) 487–494. minous shrimp Oplophorus gracilirostris: cDNA cloning of a novel imidazopyrazinone [48] S.F. Altschul, W. Gish, W. Miller, E.W. Myers, D.J. Lipman, Basic local alignment luciferase1, FEBS Lett. 481 (2000) 19–25. search tool, J. Mol. Biol. 215 (1990) 403–410. [17] E. Suarez-Morales, The mesopelagic copepod Gaussia princeps (Scott) (Calanoida: [49] J.D. Thompson, D.G. Higgins, T.J. Gibson, CLUSTAL W: improving the sensitivity of Metridinidae) from the Western Caribbean with notes on integumental pore pat- progressive multiple sequence alignment through sequence weighting, position- terns, Zootaxa 1621 (2007) 33–44. specific gap penalties and weight matrix choice, Nucleic Acids Res. 22 (1994) [18] M. Verhaegent, T.K. Christopoulos, Recombinant Gaussia luciferase. Overexpression, 4673–4680. purification, and analytical application of a bioluminescent reporter for DNA hybrid- [50] D.K. Welsh, S.A. Kay, Bioluminescence imaging in living organisms, Curr. Opin. ization, Anal. Chem. 74 (2002) 4378–4385. Biotechnol. 16 (2005) 73–78. [19] T. Rathnayaka, M. Tawa, S. Sohya, M. Yohda, Y. Kuroda, Biophysical characterization [51] E.B.Santos,R.Yeh,J.Lee,Y.Nikhamin,B.Punzalan,K.LaPerle,S.M.Larson,M. of highly active recombinant Gaussia luciferase expressed in Escherichia coli, Sadelain, R.J. Brentjens, Sensitive in vivo imaging of T cells using a membrane- Biochim. Biophys. Acta 1804 (2010) 1902–1907. bound Gaussia princeps luciferase, Nat. Med. 15 (2009) 338–344. [20] T. Rathnayaka, M. Tawa, T. Nakamura, S. Sohya, K. Kuwajima, M. Yohda, Y. Kuroda, [52] I. Serganova, E. Moroz, J. Vider, G. Gogiberidze, M. Moroz, N. Pillarsetty, M. Solubilization and folding of a fully active recombinant Gaussia luciferase with na- Doubrovin, A. Minn, H.T. Thaler, J. Massague, J. Gelovani, R. Blasberg, Multimodality tive disulfide bonds by using a SEP-Tag, Biochim. Biophys. Acta 1814 (2011) imaging of TGFbeta signaling in breast cancer metastases, FASEB J. 23 (2009) 1775–1778. 2662–2672. [21] S. Wiles, K. Ferguson, M. Stefanidou, D.B. Young, B.D. Robertson, Alternative lucifer- [53] F. Mehraein-Ghomi, H.S. Basu, D.R. Church, F.M. Hoffmann, G. Wilding, Androgen re- ase for monitoring bacterial cells under adverse conditions, Appl. Environ. Microbiol. ceptor requires JunD as a coactivator to switch on an oxidative stress generation 71 (2005) 3427–3432. pathway in prostate cancer cells, Cancer Res. 70 (2010) 4560–4568. [22] S. Inouye, Y. Sahara, Identification of two catalytic domains in a luciferase secreted [54] K.E. Luker, L.A. Mihalko, B.T. Schmidt, S.A. Lewin, P. Ray, D. Shcherbo, D.M. by the copepod Gaussia princeps, Biochem. Biophys. Res. Commun. 365 (2008) Chudakov, G.D. Luker, In vivo imaging of ligand receptor binding with Gaussia lucif- 96–101. erase complementation, Nat. Med. 18 (2012) 172–177. [23] S. Inouye, Y. Sahara-Miura, J. Sato, R. Iimori, S. Yoshida, T. Hosoya, Expression, puri- [55] B. Enjalbert, A. Rachini, G. Vediyappan, D. Pietrella, R. Spaccapelo, A. Vecchiarelli, A.J. fication and luminescence properties of coelenterazine-utilizing luciferases from Brown, C. d'Enfert, A multifunctional, synthetic Gaussia princeps luciferase reporter Renilla, Oplophorus and Gaussia: comparison of substrate specificity for C2- for live imaging of Candida albicans infections, Infect. Immun. 77 (2009) 4847–4858. modified coelenterazines, Protein Expr. Purif. 88 (2013) 150–156. [56] S. Donat, M. Hasenberg, T. Schafer, K. Ohlsen, M. Gunzer, H. Einsele, J. Loffler, A. [24] S. Knappskog, H. Ravneberg, C. Gjerdrum, C. Trosse, B. Stern, I.F. Pryme, The level of Beilhack, S. Krappmann, Surface display of Gaussia princeps luciferase allows sensi- synthesis and secretion of Gaussia princeps luciferase in transfected CHO cells is tive fungal pathogen detection during cutaneous aspergillosis, Virulence 3 (2012) heavily dependent on the choice of signal peptide, J. Biotechnol. 128 (2007) 51–61. 705–715. [57] J. Du, Q. Tang, Y. Huang, W.E. Rodney, L. Wang, G. Liang, Development of recombi- [25] G. Cheng, R.E. Davis, An improved and secreted luciferase reporter for schistosomes, nant rabies viruses vectors with Gaussia luciferase reporter based on Chinese Mol. Biochem. Parasitol. 155 (2007) 167–171. vaccine strain CTN181, Virus Res. 160 (2011) 82–88. N. Wu et al. / Biochimica et Biophysica Acta 1854 (2015) 1392–1399 1399

[58] W. Zhu, J. Zhou, K. Qin, N. Du, L. Liu, Z. Yu, Y. Zhu, W. Tian, X. Wu, Y. Shu, A reporter [63] F. Li, J. Yu, Z. Zhang, Z. Cui, D. Wang, H. Wei, X.E. Zhang, Buffer enhanced biolumines- system for assaying influenza virus RNP functionality based on secreted Gaussia lu- cence resonance energy transfer sensor based on Gaussia luciferase for in vitro de- ciferase activity, Virol. J. 8 (2011) 29. tection of protease, Anal. Chim. Acta 724 (2012) 104–110. [59] T. Wurdinger, C. Badr, L. Pike, R. de Kleine, R. Weissleder, X.O. Breakefield, B.A. [64] O. Olsson, A. Escher, G. Sandberg, J. Schell, C. Koncz, A.A. Szalay, Engineering of mo- Tannous, A secreted luciferase for ex vivo monitoring of in vivo processes, Nat. nomeric bacterial luciferases by fusion of luxA and luxB genes in Vibrio harveyi, Gene Methods 5 (2008) 171–173. 81 (1989) 335–347. [60] I. Remy, S.W. Michnick, A highly sensitive protein–protein interaction assay based [65] Y. Ohmiya, F.I. Tsuji, Mutagenesis of firefly luciferase shows that cysteine residues on Gaussia luciferase, Nat. Methods 3 (2006) 977–979. are not required for bioluminescence activity, FEBS Lett. 404 (1997) 115–117. [61] J.Y. Lee, S. Kim, W. Hwang, do, J.M. Jeong, J.K. Chung, M.C. Lee, D.S. Lee, Devel- [66] A.M. Loening, T.D. Fenn, A.M. Wu, S.S. Gambhir, Consensus guided mutagenesis of opment of a dual-luciferase reporter system for in vivo visualization of Renilla luciferase yields enhanced stability and light output, Protein Eng. Des. Sel. MicroRNA biogenesis and posttranscriptional regulation, J. Nucl. Med. 49 19 (2006) 391–400. (2008) 285–294. [67] J. Kyte, R.F. Doolittle, A simple method for displaying the hydropathic character of a [62] E. Michelini, L. Cevenini, L. Mezzanotte, D. Ablamsky, T. Southworth, B. Branchini, A. protein, J. Mol. Biol. 157 (1982) 105–132. Roda, Spectral-resolved gene technology for multiplexed bioluminescence and [68] K. Tamura, G. Stecher, D. Peterson, A. Filipski, S. Kumar, MEGA6: molecular evolu- high-content screening, Anal. Chem. 80 (2008) 260–267. tionary genetics analysis version 6.0, Mol. Biol. Evol. 30 (2013) 2725–2729.