Improving bioluminescence of a minimal luciferase by adding a charged oligopeptide: picALuc2.0

Luciferases are widely used as reporter proteins in diverse fields from basic biology to medical and environmental researches. Development of luciferase applications for reporter proteins requires small size without target inhibition, appropriate genomic insertion for high expression level, and bright emission for detection sensitivity. We previously developed the minimal luciferase picALuc, but its luminescence was still dim compared to other bright luciferases in terms of expression in Escherichia coli. In this study, diverse additions of oligopeptides with charged residues (eight amino acids in total) to the C‐terminus of picALuc enhanced luminescence by up to approximately 50‐fold, that is, enhanced enzymatic activity. Moreover, these high luminescence activities were achieved in bacterial and mammalian expression, suggesting their further applicability in many expression systems. The finding in this study that the simple addition of oligopeptides with charged residues (or charge engineering of this kind) enhances enzymatic activity may be applied to a wide variety of enzymatic reactions and protein functions.

F I G U R E 1 Model structure of picALuc fused with a charged oligopeptide and the luminescence. (A) Model structure of picALuc fused with TEVcs (ENLYFQS) and Lys (K) residues. This model structure was created by using ColabFold (AlphaFold-based). [20] picALuc, TEVcs, and K are represented by pink (ribbon), green (ribbon), and blue (sphere), respectively. (B) Luminescence of picALuc with TEVcs and K, picALuc with TEVcs and D, and WT are depicted in blue, orange and gray, respectively. Enzyme: 320 nM, substrate (CTZh): 0-125 nM, and error bars: ± 1 standard deviation (n = 3). There are significant differences between all pairs of picALuc, picALuc-ENLYFQSD, and picALuc-ENLYFQSK at each concentration: p < 0.01 (t-test) (except for the concentration of 0.00125 nM).
reporter assays, and so on. [10] In general, the small size of luciferases has the merit of not affecting their targets. In addition to the discovery and identification of luciferases from natural luminous organisms as described above, the developments of artificially designed luciferases have also recently been highly active. Several series of artificial luciferases (ALucs, ∼21 kDa) extracted from the consensus sequences of copepod luciferases have been developed and applied to colortunable bioluminescence imaging. [11,12] In addition, NanoLuc (18 kDa) developed from a deep sea shrimp would be currently one of the most used luciferases. [13,14] NanoLuc is over 150-fold brighter than FLuc and RLuc, and is applied in diverse fields such as protein-protein interactions, gene regulation, biosensors due to its brightness and ease of handling. Moreover, there is another synthetic luciferase, the tiny Tur-boLuc (16 kDa), which is used in applications such as high-throughput screening, taking advantage of its small size. [15] More recently, we developed the smallest luciferase, picALuc (13 kDa), with bright luminescence. [16] picALuc showed the same level of luminescence as ALuc and NanoLuc (above) when expressed in Cos-7 cells. In addition, it has excellent thermal stability and emits bright light from room temperature up to 80 • C. Furthermore, its BRET applications that fused the protease cleavage sequence (cs) and fluorescent protein (picALuc + TEVcs-YFP or TRBcs-mCherry2, TEV: tobacco etch virus and TRB: thrombin) demonstrated higher BRET signals than NanoLuc due to the smaller size of picALuc. However, it is inferior to ALuc and NanoLuc in terms of expression in Escherichia coli and further improvement in luminescence is desired for easier use.
We have attempted to develop sensors such as ON-type switches (protein functions such as protease treatment increase luminescence due to elimination of inhibition) and OFF-type switches (protein treatments decrease luminescence for some reasons) using protein functions. Owing to its small size and thermostability, the development of micro (ultraminiature) sensors using picALuc is highly expected to be applied in diverse fields. In the course of these developments, we expected that fusing oligopeptides consisting of TEV protease cleavage sequence (TEVcs, ENLYFQS) (used in the previous study for the BRET application [16] ) with charged residues (K or D) to the Cterminus of picALuc might promote intraenzymatic stabilization or regulate luminescence reaction (Figure 1), because a variety of electrostatic interactions (or charge engineering) have been utilized in protein structure and function. [17][18][19] Unexpectedly, an increase in luminescence was observed when the reaction was driven by the addition of substrate CTZh.
In this study, we investigated the luminescence properties of picALuc fused with charged oligopeptides at its C-terminus. As expected, when the TEVcs and K(+) or D(-) were added, these oligopeptides-fused enzymes emitted brighter luminescence than the original (wild type) one. On the other hand, only slight enhancements in luminescence were observed when polyalanines or GS linkers of different lengths with charged residues (K or D) were added. Furthermore, when we examined single-residue mutations to Ala within TEVcs, Ala mutations at a specific position resulted in high luminescence in oligopeptides with charged residues (K, D, and E) in common. Notably, the addition of an oligopeptide with Ala at this position and K at the end enhanced the luminescence by approximately 50-fold.

Plasmid construction
To yield plasmids encoding picALuc-ENLYFQSP-Lys/Asp, PCR was performed with the template, pet-32 vector encoding picALuc1.0 [16]   The protein added with 15% of glycerol was stored at 4 • C or −80 • C before use.

Lysate preparation
The cells expressing picALuc1.0 or the mutants were harvested by centrifugation, and the cytoplasmic fraction was recovered. The fraction was suspended with B-PER Bacterial Protein Extraction Reagent (Thermo Fisher Scientific, MA, USA), and rotated gently for 15 min.
at room temperature. The suspension was centrifuged, and the supernatant was collected as the lysate. The concentrations of lysates were compared from the band intensities of electrophoresis using Mini-PROTEAN-TGX Stain Free gel, and the lysates were adjusted to the same concentration to evaluate the luminescence intensities by the calculation using GelDoc system.

Improvement of luminescence of picALuc by oligopeptide fusions with charged residues at terminus
Oligopeptides with a protease cleavage sequence and charged residues, (i) TEVcs + positively charged residue (ENLYFQSK) and (ii) TEVcs + negatively charged residue (ENLYFQSD), were fused to the C-terminus of picALuc as follows, and their luminescence were compared with that of wild type (WT). First of all, we aimed to achieve sufficient light even with a small amount of substrate

Effects of single-residue mutations to alanine in the oligopeptide region, and different-length polyalanines and GS linkers
In the fused oligopeptides above, the sequence ENLYFQS, excluding the charged residues, was based on TEVcs. To identify more opti-  (Figure 2, right). In particular, those with the terminal K did not significantly depend on the length, while those with the terminal D tended to decay (from 0.4 to 0.1 × 10 8 s −1 ) in proportion to their length, except for the longest 9 aa Ala oligopeptide with low luminescence. In the case of GS linkers of different lengths, the luminescence was as low as 0.1 × 10 8 s −1 . For comparison with WT, we examined the luminescence of these oligopeptides with GS linkers of different lengths and the terminal D ( Figure S2). As a result, the luminescence of WT was between those of 7 aa GS linker + D and 3 aa GS linker + D. Thus, from these results, it was found that not only the four mutants with high luminescence obtained above, but also most of the mutants depicted in Figure 2 (single-residue mutations to Ala and polyalanines) exhibited higher luminescence than WT.

Comparison of additions of differently charged residues at the terminus and effect of single-residue mutations to alanine in the E-added case
Among the protein-constituting amino acids, there are negatively charged residues, D and E, and positively charged residues, K and R (except for protonated His(H)). We compared luminescence when these differently charged residues (D, E, K, and R) and a neutral alanine F I G U R E 3 Comparison of differently charged residues at the terminus of picALuc-TEVcs+X. Luminescence of wild type picALuc (WT) and picALuc with oligopeptides of TEVcs+D/E/K/R/A and are depicted in gray, orange, pink, blue, cyan, and green, respectively. Enzyme: 500 pM, substrate (CTZh): 25 μM, and error bars: ± 1 standard deviation (n = 3). There are significant differences between all pairs of WT, D, E, K, R, and A: p < 0.05 (t-test).
(A) were added to the TEVcs sequence in the purified fusion-proteins.
As a result, the luminescence of TEVcs+A was comparable to that of WT, and all oligopeptides with charged residues gave higher luminescence than WT (Figure 3). In addition, in this comparison of charged residues using TEVcs, not only TEVcs+K but also TEVcs+E showed higher luminescence than others, then the single-residue mutations to Ala were also performed in the case of TEVcs+E. When each lysate of TEVcs+E with single mutation to Ala was reacted with the substrate, the highest luminescence was obtained again with the oligopeptide with Ala introduced at the 7 th position in TEVcs sequence (the oligopeptide with Ala introduced at the 4 th position also showed moderately high luminescence) ( Figure 2B). Based on this result that the oligopeptide with Ala introduced at the 7 th position in TEVcs+E showed high luminescence and the results of the four high luminescence oligopeptides obtained in Figure 3 above, it was found that all of TEVcs+K, TEVcs+D and TEVcs+E showed high luminescence with introduction of Ala at the 7 th position in common. In other words, the combination of TEVcs(7 th pos: A) + charged residues exhibited high luminescence.

Oligopeptide fusions (with Ala at the 7 th position and differently charged residues) to picALuc
From the above results, oligopeptides with Ala at the 7 th position in TEVcs showed high luminescence. Therefore, we compared the luminescence of oligopeptides (7 th pos: A) with differently charged residues at the terminus. As a result, all four mutants (picALuc-TEVcs(7 th pos:  Figure 4B). We named this brightest mutant "picALuc2.0." In our previous study, [16] Cos-7 cell-expressed picALuc1.0 showed comparable luminescence to ALuc and NanoLuc, but E. coli-expressed picALuc1.0 showed inferior luminescence. Therefore, the 25-to 50-fold improvement in luminescence of picALuc2.0 in E. coli expression obtained in this study would be expected to be even more useful for a variety of applications.

Bioluminescence upon secreted expression in Cos-7 cells
In order to explore the possibility of further applications of picALuc2.0 toward its use as a reporter protein, the bioluminescence of picALuc2.0 upon secretory expression in mammalian Cos-7 cells was measured ( Figure 5). Moreover, to validate the luminescence enhancement observed above, the bioluminescence of picALuc1.0 was also measured. As a result, the luminescence intensity of picALuc2.0 was 8.47 with other marine copepod luciferases such as GLuc and ALuc [9,11] (in the development of the original picALuc1.0, we utilized these disulfide bonds [16] ). The presence of disulfide bonds is one of the important features of secreted luciferases, and the secreted luciferases have attracted great interests for bioimaging. [9,10,21] From the results observed here, it would be expected to further expand the applicability using secreted luciferases

DISCUSSION
In this study, we achieved enhanced luminescence (25-50-fold) by fusing a charged oligopeptide to the C-terminus of the previous version of picALuc, that is, picALuc1.0 ( Figure 4). Previous picALuc1.0 [16] has a molecular weight of 13 kDa, but picALuc-TEVcs(7th pos: A)+K (i.e., picALuc2.0) with the highest luminescence value here corresponds to 14 kDa. In picALuc2.0, the 7 th protease-treated sequence (ENLYFQS) is mutated to Ala, and this ENLYFQA can also be treated with TEV protease. The TEV cleavage sequence is commonly expressed as EXLYΦQ∖Φ, where X is any residue, Φ is any large or middle hydrophobic residue, and Φ is any small hydrophobic or polar residue. [22,23]  based ColabFold [20] is depicted in Figure 1A. picALuc is derived from ALuc, an artificial luciferase, and ALuc is homologous to GLuc. [16] GLuc was analyzed for NMR structures and found to be very flexible. [24] In addition, the GLuc structure was one of the difficult targets to predict in the previous protein structure prediction contest (CASP14 (2020)). [25] Therefore, it might be difficult to analyze the picALuc structure, which is a miniaturized ALuc, its structure attached with a charged oligopeptide (picALuc2.0), and its substrate-bound structure.
We are currently working on structural analysis of these structures using various methods.
Charged residues are presumed to significantly affect the reactivity and stability of enzymes. In this study, the addition of a charged residue to the end of an oligopeptide linked to the luciferase picALuc promoted its stabilization and enhanced luminescence. Cather containing a lysine residue (K(+)) to form an intermolecular isopeptide bond between the pairs, that is, a set of negatively and positively charged residues (D and K, respectively) is utilized. [26] In this study, based on the TEVcs used in our previous work, we demonstrated that luminescence was enhanced by adding a charged residue to the end of an enzyme and oligopeptide. In protein studies, the cleavage sequences are utilized when protease treatment is applied, but various tags are also used to identify protein expression, and so on. Therefore, the simple idea of adding charged residues to oligopeptides could be utilized for a wide variety of protein studies.
The cause of enhanced luminescence observed in this study may be related to the increased affinity of enzyme. In the picALuc- leading to a change in affinity. Moreover, the blue-shifted peaks in the measured emission spectra may be due to different emitting species ( Figure S1): Neutral form and amide, phenolate, and pyrazine anions are known to emit short to long wavelengths. [27] Further structural and luminescence investigations would be required to comprehend these relationships.
In summary, we have enhanced luminescence up to approxi-

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request. The picALuc2.0 plasmid is available freely to academic researchers under appropriate agreement with Shimadzu Corporation upon request.