Luciferase oluc mutant and use thereof
By modifying the OLuc mutant of luciferase, the problem of instability of luciferase at high temperatures was solved, achieving high yield and high thermal stability, making it suitable for large-scale production and industrial applications.
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- BIORTUS WUXI CO LTD
- Filing Date
- 2025-11-20
- Publication Date
- 2026-07-09
AI Technical Summary
Existing luciferases are unstable at high temperatures, which limits their application in fields such as bioimaging, medical diagnostics, and molecular biology, especially in environments with high temperature requirements.
By modifying the wild-type luciferase OLuc from deep-sea shrimp, a luciferase OLuc mutant was obtained. The modified amino acid sequence is shown in SEQ ID NO.2. The mutant was constructed on the pET-28a expression vector and expressed in Escherichia coli. After purification, a high-yield, high-thermal-stability luciferase OLuc mutant was obtained.
The luciferase OLuc mutant exhibits high thermal stability at 85.30℃ and increases protein yield by 2.5 times, making it suitable for large-scale production, reducing application costs, and broadening its application scope.
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Figure CN2025136359_09072026_PF_FP_ABST
Abstract
Description
A luciferase OLuc mutant and its application Technical Field
[0001] This invention relates to the field of bioengineering technology, specifically to a luciferase OLuc mutant and its applications. Background Technology
[0002] Luciferases are a class of enzymes that catalyze the emission of luciferin, and are widely found in various organisms, especially bioluminescent organisms (such as fireflies and deep-sea creatures). The most famous representative of this enzyme is the luciferase from fireflies, which catalyzes the reaction of luciferin with oxygen, ATP, and divalent magnesium ions to produce visible light (fluorescence) under specific conditions. In this process, the emitted light typically has a relatively long wavelength (such as blue or green light), and the intensity of the light is closely related to the concentration of the reactants. Therefore, the light signal produced by luciferase-catalyzed reactions has the characteristics of high sensitivity, low background noise, and quantifiability, making it an ideal choice for molecular probes and sensors, and it has been widely used in cell and molecular biology research. Luciferase reporter gene technology has become an important tool in the study of gene expression, protein-protein interactions, and drug screening. Furthermore, luciferase also has significant application prospects in medical diagnostics, cell imaging, and animal model research.
[0003] In some applications, such as bioimaging or in vivo imaging, traditional luciferases typically experience a decrease in activity or become ineffective at high temperatures, which is detrimental to long-term monitoring of biological responses, lesions, and drug effects in vivo. Furthermore, in some molecular biology experiments or environmental monitoring processes, where high temperature requirements are necessary, the poor thermal stability of natural luciferases limits their application. With technological advancements, scientists have developed improved luciferases through genetic engineering and directed evolution. These improvements have significantly optimized the performance of luciferases, resulting in substantial enhancements in luminescence efficiency, spectral characteristics, and stability. These novel luciferases have further broadened their application range across various fields.
[0004] The small subunit of luciferase derived from deep-sea shrimp was identified as a luciferase in 2000. After the discovery of natural luciferase, many researchers have modified the enzyme to develop enzymes with higher activity and a wider range of applicable pH (6-9). However, the thermostability of the enzyme has not been greatly improved, so the enzyme is still limited in some applications. Summary of the Invention
[0005] The purpose of this invention is to provide a luciferase OLuc mutant and its applications in order to expand the application conditions of luciferase OLuc and make it suitable for large-scale industrial use.
[0006] The present invention achieves the above objectives through the following technical solutions:
[0007] As a first aspect of the present invention, a luciferase OLuc mutant is provided, which is obtained by modifying the amino acid sequence of wild-type luciferase OLuc, and the amino acid sequence of the luciferase OLuc mutant is shown in SEQ ID NO.2.
[0008] As a second aspect of the present invention, a polynucleotide is provided that encodes the luciferase OLuc mutant as described above, the polynucleotide sequence encoding the luciferase OLuc mutant being shown in SEQ ID NO.3.
[0009] As a third aspect of the present invention, a recombinant plasmid is provided, the recombinant plasmid being an expression vector containing the polynucleotide sequence as described above and capable of correspondingly translating and expressing the luciferase OLuc mutant as described above.
[0010] A further improvement is that the expression vector is pET-28a.
[0011] As a fourth aspect of the present invention, an expression system for a luciferase OLuc mutant is provided, comprising BL21(DE3) cells containing any of the recombinant plasmids described above or whose genomes integrate the polynucleotides described above.
[0012] As a fifth aspect of the present invention, a method for preparing a luciferase OLuc mutant as described in the first aspect is provided, characterized by comprising the following steps:
[0013] (1) Gene sequence encoding the amino acid sequence of the luciferase OLuc mutant was synthesized based on the amino acid sequence of the mutant, and then the gene sequence was constructed on an expression vector to obtain a recombinant plasmid.
[0014] (2) The recombinant plasmid obtained in step (1) is expressed using the Escherichia coli prokaryotic expression system to obtain the expression product. The expression product is then purified to obtain the luciferase OLuc mutant.
[0015] As a sixth aspect of the present invention, an application is provided of the luciferase OLuc mutant as described above in improving the yield or thermal stability of luciferase OLuc.
[0016] As a seventh aspect of the present invention, an application of the luciferase OLuc mutant as described above in molecular biological detection, food detection, environmental detection, or medical detection is provided.
[0017] The present invention has the following beneficial effects:
[0018] This invention modifies the wild-type luciferase OLuc derived from deep-sea shrimp to obtain a luciferase OLuc mutant. The modified luciferase OLuc mutant exhibits a Tm value of 85.30℃, demonstrating very high thermal stability. The Tm value of the luciferase OLuc mutant is 18.17℃ higher than that of the previously reported luciferase NanoLuc, and it still retains high luciferase activity. This overcomes the problem of enzyme instability at high temperatures that limits its application, providing better application scenarios for storage and industrial production, thus broadening its application scope. Furthermore, the yield of the luciferase OLuc mutant in *E. coli* reaches nearly 90 mg / L, which is 2.5 times higher than that of luciferase NanoLuc. This high yield makes it more suitable for large-scale production and reduces costs in subsequent application development. Attached Figure Description
[0019] Figure 1 shows the low-level expression results of the OLuc mutant and the control luciferase Nanoluc provided by the present invention.
[0020] Figure 2 shows the affinity purification results of the OLuc mutant luciferase and the control luciferase Nanoluc provided by the present invention.
[0021] Figure 3 shows the activity identification and detection results of the OLuc mutant luciferase and the control luciferase Nanoluc provided by the present invention.
[0022] Figure 4 shows the thermostability test results of the OLuc mutant luciferase and the control luciferase Nanoluc provided by this invention. Detailed Implementation
[0023] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.
[0024] 1. Materials and Reagents
[0025] Unless otherwise specified, all methods used in this invention are conventional methods known to those skilled in the art. Where specific conditions are not specified, they shall be performed according to conventional conditions or conditions recommended by the manufacturer. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.
[0026] 2. Method
[0027] 2.1 Construction of recombinant luciferase plasmid
[0028] The gene sequences of the recombinant luciferase provided in this invention are all obtained through gene synthesis.
[0029] The wild-type luciferase OLuc is derived from the deep-sea shrimp Oplophorus gracilirostris. The amino acid sequence of the wild-type luciferase OLuc is shown in SEQ ID NO.1. The signal peptide sequence at positions 1-27 of the N-terminus was truncated during recombinant expression ("MAYSTLFIIALTAVVTQASSTQKSNLT").
[0030] Mutation design was performed on the wild-type luciferase OLuc with the signal peptide sequence removed to obtain the luciferase OLuc mutant. The amino acid sequence of the luciferase OLuc mutant is shown in SEQ ID NO.2, and the corresponding gene sequence is shown in SEQ ID NO.3.
[0031] To better compare the performance of the modified luciferase, this invention also compared it with Nanoluc, a luciferase with PDB ID 7SNS that exhibits higher activity than the wild-type luciferase OLuc. The amino acid sequence of Nanoluc is shown in SEQ ID NO. 4. This enzyme was obtained by modifying the wild-type luciferase OLuc, and its reported activity is approximately 9 times higher than that of the wild-type luciferase OLuc. (See Tomabechi, Yuri et al. "Crystal structure of nanoKAZ: The mutated 19kDa component of Oplophorus luciferase catalyzing the bioluminescent reaction with coelenterazine.").
[0032] To facilitate protein purification, the tag 8His and the restriction site 3C were added to the N-terminus of the sequences shown in SEQ ID NO.2 and SEQ ID NO.4 (the amino acid sequence of 8His is “HHHHHHHH”, and the amino acid sequence of 3C is “LEVLFQGP”).
[0033] All luciferase genes were constructed on the expression vector pET-28a after synthesis, and all recombinant plasmids were sequenced and verified to be completely consistent with the target sequence.
[0034] 2.2 Protein Expression of Recombinant Luciferase
[0035] 2.2.1 Low-level expression of recombinant luciferase
[0036] Using standard molecular biology techniques, the recombinant plasmids of the constructed luciferase OLuc mutant and the control luciferase Nanoluc were transformed into BL21(DE3) competent E. coli cells in a clean bench and cultured overnight at 37°C. Single colonies from the overnight culture were picked and transferred to 5 ml of LB broth and incubated at 37°C until the bacterial culture OD... 600 When the pH is 0.6-0.8, a small amount of bacterial culture is fixed with loading buffer, and a small amount of bacterial culture is added to glycerol and frozen to -80℃. The remaining bacterial culture is added to 0.5mM IPTG and induced at 15℃ for 16 hours. The bacterial cells are then collected and the induced bacterial culture is analyzed by SDS-PAGE.
[0037] The results are shown in Figure 1. Both the OLuc mutant and the control luciferase Nanoluc were significantly expressed in BL21(DE3) Escherichia coli and had good protein solubility.
[0038] 2.2.2 High-level expression of recombinant luciferase
[0039] The strains exhibiting the above-mentioned clear expression were inoculated into 50 ml of LB liquid medium and cultured overnight at 37°C. The bacteria cultured overnight were then inoculated into 1 L of LB liquid medium at a ratio of 1:100 and cultured at 37°C until the bacterial culture reached OD. 600 When the bacterial concentration is 0.6-0.8, add 0.5 mM IPTG, incubate overnight at 15°C, and collect the bacterial cells by centrifugation at 5000 rpm.
[0040] 2.3 Protein purification of recombinant luciferase
[0041] The collected bacterial cells were weighed and added to a 1:10 ratio with the appropriate volume of lysis buffer (50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 5% glycerol). The cells were homogenized using an autoclave, and the supernatant was collected by centrifugation at 16,000 rpm. All recombinant luciferases had an 8His tag, and the proteins were enriched and purified using a Ni Bestarose FF affinity chromatography column. The specific procedure was as follows: the Ni Bestarose FF affinity chromatography column was first equilibrated with lysis buffer to 10 column volumes. Then, the lysis supernatant was loaded onto the Ni Bestarose FF affinity chromatography column, and eluted with imidazole solutions of different gradients. The proteins eluted with different gradients of imidazole were collected for SDS-PAGE analysis, and the protein concentration was determined using Nanodrop to calculate the protein yield.
[0042] The protein purification results are shown in Figure 2. A luciferase OLuc mutant and a control luciferase Nanoluc were obtained with good purity. According to Nanodrop assay results, the protein yield of the modified luciferase OLuc mutant was approximately 90 mg / L, while the protein yield of the control luciferase Nanoluc was approximately 35 mg / L. The yield comparison shows that the yield of the modified luciferase OLuc mutant was approximately 2.5 times higher than that of the control luciferase Nanoluc.
[0043] 2.4 Activity Assay of Recombinant Luciferase
[0044] Luciferase binds to its substrate, releasing a chemiluminescent signal that can be detected. Higher luciferase activity results in a greater number of photons produced per unit time during the substrate reaction, leading to a stronger detected light signal. Enzyme activity is expressed as the light signal intensity absorbed per nanomolar of protein.
[0045] The specific procedures for the luciferase activity assay are as follows:
[0046] Furimazine (GLPBiO, catalog number: PBI1939) was prepared as a 1 mM stock solution and aliquoted for use. The luciferase activity assay buffer was 50 mM Tris-HCl (pH 8.0). The Furimazine concentration was diluted to 2 μM with the buffer, and the luciferase was serially diluted 2-fold from 100 nM to a total of 12 concentrations. 10 μL of the substrate was transferred to a 384-well plate with two replicates. 10 μL of the luciferase assay was transferred to the corresponding well, immediately centrifuged, and vortexed to mix. The fluorescence signal values generated by the reaction were collected using a TECANF200 microplate reader. Data analysis was performed using Graph Pad Prism9 software to obtain the enzyme activity parameters of the tested proteases. Figure 3 shows the enzyme activity parameters of different luciferases obtained by Graph Pad Prism9 software analysis.
[0047] The luciferase activity results are shown in Figure 3. The activity of the modified luciferase OLuc mutant was 4,314,173 (RFU / nM), while the activity of the control luciferase Nanoluc was 8,219,667 (RFU / nM). The activity of the modified luciferase mutant was approximately 1.9 times lower than that of the control luciferase Nanoluc. However, according to previous reports, the activity of NanoLuc was approximately 9 times higher than that of the wild-type OLuc. This indicates that the luciferase OLuc mutant provided by this invention still possesses good enzyme activity.
[0048] 2.5 Thermostability Test of Recombinant Luciferase
[0049] The thermostability assay for recombinant luciferase uses protein thermal shuffling (ThermoFluor). Utilizing the structural characteristics of proteins, proteins possess hydrophobic regions hidden internally. As temperature rises, this structure opens up, exposing the hydrophobic regions. The fluorescent dye SYPRO Orange can then bind to these regions, stimulating fluorescence. A melting curve is formed based on the change in fluorescence intensity. The temperature corresponding to the maximum derivative of the melting curve is the melting point (Tm). The more stable the protein, the higher the measured Tm value.
[0050] The specific procedure for determining the thermostability of luciferase is as follows:
[0051] Add 5 μg of luciferase to each well of a 96-well PCR plate, and then add 10×SYPRO Orange (ThermoFisher Sciientific, S6650) fluorescent dye to the corresponding wells. Place the 96-well PCR plate in a qPCR instrument, set the instrument parameters, and increase the temperature from 20℃ to 99℃ at a gradient of 1℃ per minute. Calculate the protein melting curve.
[0052] As shown in Figure 4, the Tm value of the modified luciferase OLuc mutant was 85.30℃, while the Tm value of the control luciferase Nanoluc was 67.13℃. The modified luciferase OLuc mutant exhibited significantly improved thermal stability, reaching over 85℃, an increase of 18.17℃ compared to Nanoluc. The modified luciferase OLuc mutant possesses high thermal stability, overcoming the problem of enzyme instability at high temperatures that limits its application, and providing better application scenarios for its storage and industrial production.
[0053] 3. Conclusion
[0054] The above description shows that the luciferase OLuc mutant provided by the present invention has the characteristics of high protein yield, high thermal stability, and good enzyme activity, which has a wider range of application conditions and stronger practical application value, and is more suitable for large-scale production and industrial use.
[0055] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.
[0056] The sequence involved in this invention:
[0057] Serial Number: 1
[0058] Source: Synthetic construct wild-type OLuc amino acid sequence
[0059] Serial Number: 2
[0060] Source: Synthetic construct luciferase OLuc mutant protein sequence
[0061] Serial Number: 3
[0062] Source: Synthetic construct luciferase OLuc mutant nucleotide sequence
[0063] Serial Number: 4
[0064] Source: Synthetic construct control luciferase Nanoluc PDB:7SNS
Claims
1. A luciferase OLuc mutant, characterized in that, The luciferase OLuc mutant was obtained by modifying the amino acid sequence of wild-type luciferase OLuc, and the amino acid sequence of the luciferase OLuc mutant is shown in SEQ ID NO.
2.
2. A polynucleotide, characterized in that, The polynucleotide encodes the luciferase OLuc mutant as described in claim 1, and the polynucleotide sequence encoding the luciferase OLuc mutant is shown in SEQ ID NO.
3.
3. A recombinant plasmid, characterized in that, The recombinant plasmid is an expression vector containing the polynucleotide sequence as described in claim 2 and capable of correspondingly translating and expressing the luciferase OLuc mutant as described in claim 1.
4. The recombinant plasmid according to claim 3, characterized in that, The expression vector is pET-28a.
5. An expression system for a luciferase OLuc mutant, characterized in that, The BL21(DE3) cells contain the recombinant plasmid as described in any one of claims 3-4 or whose genome integrates the polynucleotide as described in claim 2.
6. A method for preparing the luciferase OLuc mutant as described in claim 1, characterized in that, Includes the following steps: (1) Based on the amino acid sequence of the luciferase OLuc mutant as shown in SEQ ID NO.2, a gene sequence that can encode its amino acid sequence is synthesized, and the gene sequence is then constructed on an expression vector to obtain a recombinant plasmid. (2) The recombinant plasmid obtained in step (1) is expressed using the Escherichia coli prokaryotic expression system to obtain the expression product. The expression product is then purified to obtain the luciferase OLuc mutant.
7. The application of the luciferase OLuc mutant as described in claim 1 in improving the yield or thermal stability of luciferase OLuc.
8. The application of the luciferase OLuc mutant as described in claim 1 in molecular biological detection, food detection, environmental detection, or medical detection.