2.1.

Homology Modeling

Homology modeling of Eluc was conducted using SWISS-MODEL.¹⁵ The three-dimensional (3-D) structure of a substrate binding luciferase derived from Luciola cruciata (PDB: 2D1R) was used as a template. Amino acid sequences of click beetle green (CBG) (AY258595.1) and click beetle red (CBR) (AAP83303.1) were obtained from GenBank.

2.2.

Site-Directed Mutagenesis

The cDNA for Eluc (Emerald Luc®) was purchased from TOYOBO (Shiga, Japan). Mutations at R214, H241, S246, and H347 of Eluc were introduced by site-directed mutagenesis with the primers shown in Table 1 and their complementary primers. Template DNA (approx. $0.1\mu \mathrm{mol}$) was added to a polymerase chain reaction (PCR) mixture containing $0.8\mu \mathrm{M}$ 5’-phosphorylated primer, 0.2 mM dNTP mix, $5\times$ PrimeSTAR Buffer, and 2.5 U of PrimeSTAR HS DNA Polymerase (Takara Bio Inc.). The PCR cycling parameters were the following: the solution was heated at 98°C for 2 min, followed by 30 cycles of 10 s at 98°C, 5 s at 55°C, and 7 min at 72°C. The mixture was treated with DpnI (10 U; Takara Bio Inc.) at 37°C for 4 h. The introductions of the mutations were verified using DNA sequencing (Eurofins Sequencing Service).

Table 1

Mutagenic primers. DNA sequences of the mutagenic primers are listed. The mutations are shown as italics.

2.3.

Expression and Purification of Luciferases

Wild-type and mutant luciferases were expressed in E. coli BL21. The E. coli harboring the expression plasmid was grown in 1 mL of LB medium at 37°C until the mid-log phase (${\mathrm{O.}\mathrm{D.}}_{600}=0.4–0.6$). Then 1 mM IPTG was added and the cultures were incubated at 16°C for 24 h. The cells were collected by centrifugation at 4°C and were suspended in $30\mu \mathrm{L}$ of phosphate buffered saline (PBS) with subsequent sonication for 10 min at medium intensity using Bio Ruptor (Cosmo Bio Co. Ltd., Tokyo, Japan). The lysate was centrifuged to obtain the soluble fraction containing the luciferase. The solution was loaded onto equilibrated His SpinTrap (GE Healthcare) and was centrifuged at 4°C for 2 min. The column was washed several times with $300\mu \mathrm{L}$ of PBS containing 30 mM imidazole until the absorbance of the flowthrough solution at 280 nm became negligible. The protein was eluted with $200\mu \mathrm{L}$ of 300 mM imidazole in PBS and was stored on ice until measurements of bioluminescence were conducted.

2.4.

Luminescence Measurement

First, $5\mu \mathrm{L}$ of the crude or purified luciferase solution was added to $45\mu \mathrm{L}$ of reaction mix containing $400\mu \mathrm{M}$ luciferin, 1 mM adenosine triphosphate (ATP), 5 mM ${\mathrm{MgSO}}_4$, and 30 mM HEPES ($\mathrm{pH}=7.6$). Bioluminescence spectra were measured using a spectroluminometer (AB-1850 LumiFlSpectroCapture; Atto Corp., Tokyo, Japan) with a slit width of 0.5 mm. Background luminescence from the reaction mix without luciferases was subtracted from the obtained spectra. Luminescence was detected with the exposure time of 75 s and the intensities were evaluated by photon counts at the wavelength of the peak maximum. To obtain kinetics of luminescence, luminescence intensities were integrated every 15 s. The half-life of the luminescence was evaluated by the duration from the luminescence peak to the point when luminescence was decreased to the half of its peak intensity.

3.1.

Eluc Structures Based on Homology Modeling

According to a previous research on Ppy luciferase mutants, residues near the luciferin binding pocket affect the bioluminescence wavelength.¹⁶ We inferred that amino acid mutations around the D-luciferin binding site of Eluc induce a red-shift in the bioluminescence.¹⁷ To identify the residues around the luciferin binding site of Eluc, we performed homology modeling of Eluc using SWISS-MODEL.¹⁵ The amino acid sequence of Eluc was aligned with that of Luciola cruciata (Lcr) luciferase, of which a crystal structure incorporating a substrate analog 5’-$O$-[$N$-(dehydroluciferyl)sulfamoyl]adenosine (DLSA) has already been reported (PDB: 2D1R).¹⁸ Based on the alignment results, we constructed a predicted structure of Eluc. The structure displayed that amino acid residues of R214, H347, H241, and S246 are located in the binding pocket, close to D-luciferin within a distance of 6 Å. R214 and H347 are near the benzothiazole ring of the substrate, whereas H241 is close to the thiazolone ring. The residue of S246 is directed perpendicularly to the center of D-luciferin [Fig. 1(a)].

Fig. 1

(a) The luciferin binding site in the predicted structure of Emerald luciferase (Eluc). Luciferin is located in the center. Amino acid residues close to the substrate are shown in color. (b, c) Amino acid sequences of three click beetle luciferases, Eluc, CBG, and CBR. The seven residues which differ between CBG and CBR are indicated by arrows. Black arrows indicate residues conserved only in Eluc and CBG. Gray arrows indicate residues not conserved in click beetle luciferases (CBG and CBR) and in green luciferases (Eluc and CBG).

To obtain additional clues to induce a red-shift to Eluc, we compared the amino acid sequence of Eluc with CBG and CBR luciferases, both of which are derived from Pyrophorus plagiophthalamus. The amino acid residues that are conserved in Eluc and CBG, but not in CBR, are candidates for the mutation sites to generate red-shifted Eluc mutants. We identified such candidates of four amino acid residues of Eluc: V223, S246, N345, and H347 [Figs. 1(b) and 1(c)]. Taken together with the result of the homology modeling, we selected S246 and H347 as amino acid residues for mutagenesis to produce red-shifted Eluc mutants.

3.2.

Spectral Properties of Eluc Mutants at S246 and H347

Based on a comparison between the amino acid sequences of Eluc and CBR [Figs. 1(b) and 1(c)], we examined spectral properties of Eluc mutants, S246H and H347Q, in a physiological condition ($\mathrm{pH}=7.6$). The emission maxima of the bioluminescence spectra were red-shifted upon insertion of the single mutations (Fig. 2). The S246H mutation caused a red-shift of 21 nm, and the H347Q mutation showed a greater peak shift of 66 nm. The H347Q mutant showed a larger red-shift than any other single-mutated beetle luciferases already reported. The luminescence intensity of the H347Q mutant was, however, 20 times lower than that of the wild-type Eluc. To obtain a mutant with higher bioluminescence intensity, we introduced an alanine instead of a glutamine at the position of H347. The H347A mutant showed a 54-nm red-shift compared with wild-type Eluc (Fig. 2). The bioluminescence intensity was 10 times stronger than that of the H347Q mutant. We introduced a mutation of S246H into the mutant H347A, causing the emission maximum of 602 nm, which is a 64 nm longer wavelength than that of the wild type. This value was almost identical to that of the H347Q mutant. The bioluminescence intensity was not decreased by the extra mutation of S246H (Fig. 2).

Fig. 2

Wavelengths and intensities of emission maxima for the wild-type Eluc and its mutants.

3.3.

Comprehensive Mutagenesis at R214 and H241 of Eluc

We obtained an Eluc mutant with red-shifted luminescence only by insertion of S246H/H347A. However, the emission maximum of the mutant was still shorter than that of natural red luciferases such as PhRE ($\lambda \max =628\mathrm{nm}$). Then we successively inserted mutations at two other amino acid residues, R214 and H241. Previous studies of mutagenesis and modeling of Ppy and Lcr luciferases demonstrated that mutations at R214 and H241 affected bioluminescence spectra.^19–22 Based on such information, we performed systematic mutagenesis at the positions in the S246H/H347A Eluc mutant, and evaluated the properties of bioluminescence. First, the R214 residue in the S246H/H347A mutant was mutated to yield the triple mutants, and the bioluminescence spectra of each mutant were investigated. All the mutants caused red-shifted bioluminescence [Fig. 3(a)], of which the R214M/S246H/H347A mutant showed the most red-shifted emission ($\lambda \max$ was 620 nm). The intensity of this mutant was, however, low (data not shown). Therefore, we further systematically introduced mutations at H241 in the R214K/S246H/H347A mutant instead of the R214M/S246H/H347A mutant to prepare the 19 mutants. In these mutants, luminescence of aspartic acid, glutamic acid, and isoleucine mutants was not detectable [Figs. 3(b) and 3(c)]. Mutations of H241K in the R214K/S246H/H347A Eluc mutant showed the largest wavelength shift; the mutant emitted 7-nm longer wavelength light than that of the R214K/S246H/H347A mutant. The luminescence properties of the wild type and its mutant luciferases are summarized in Table 2 and Fig. 3(d). The luminescence of mutant luciferases showed transient increases in a few minutes with gradual decreases over 15 min thereafter, whose property is almost the same as that of the wild-type luciferase (Fig. 4). Consequently, we developed an 88-nm red-shift Eluc mutant by insertion of four amino acid mutations (R214K, H241K, S246H, and H347A) into the wild-type Eluc [Fig. 3(d)].

Fig. 3

(a,b) Emission maxima of the bioluminescence for R214X/S246H/H347A (a) and H241X/R214K/S246H/H347A (b) mutants. Each column indicates a single mutant with a mutation at R214 (a) or H241 (b). Negatively and positively charged residues are colored, respectively, in black and white. Hydrophobic residues are shown in gray. Asp, Glu, Ile mutants for R214K/H241X/S246H/H347A showed no detectable bioluminescence. (c) Wavelengths and intensities of emission maxima for the R214K/H241X/S246H/H347A mutants. The concentration of luciferases was six times higher than the condition of the experiment shown in Fig. 2. (d) Bioluminescence spectra of wild-type Eluc and its mutants are shown. Each spectrum was normalized at peak intensity.

Table 2

Luminescence properties of the wild type and mutant Emerald luciferases. Half-life was evaluated by the duration from the luminescence peak to the time point when the luminescence intensity decreased to the half of its peak intensity.

Fig. 4

Time dependence of luminescence intensities for wild type and three typical Eluc mutants. Luminescence intensities were normalized at their peak intensities.