Luciferase oLuc mutant and application thereof

Abstract

The application discloses a luciferase OLuc mutant and application thereof, relates to the technical field of bioengineering, and is based on the amino acid sequence of a wild-type luciferase (OLuc) from a deep-sea shrimp and is obtained through modification, wherein the amino acid sequence of the luciferase OLuc mutant is shown as SEQ ID NO. 2. Compared with the reported NanoLuc based on the modified OLuc, the OLuc mutant has higher thermal stability, and the Tm value has reached above 85 DEG C, which is 18 DEG C higher than that of the NanoLuc, so that the problem of instability of the enzyme at high temperature and the limitation of application of the enzyme can be overcome, application scenarios for storage and better application of the enzyme in industrial production are provided, and the enzyme has wider application conditions. In addition, high yield of the enzyme makes the enzyme more suitable for large-scale production and reduces the cost in subsequent application development.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a luciferase OLuc mutant and applications thereof. Background Art

[0002] Luciferases are a class of enzymes that catalyze the production of luciferin. They are found in a wide variety of organisms, particularly bioluminescent organisms such as fireflies and deep-sea creatures. The most famous example of this enzyme is the firefly luciferase, which catalyzes the reaction of luciferin with oxygen, ATP, and divalent magnesium ions, producing visible light (fluorescence) under specific conditions. The light emitted in this process typically has a long wavelength (such as blue or green), and its intensity is closely related to the concentration of the reactants. Therefore, the light signal produced by the luciferase-catalyzed reaction is highly sensitive, has low background noise, and is quantifiable, making it an ideal choice for molecular probes and sensors. It has been widely used in cell and molecular biology research. Luciferase reporter gene technology has become an important tool for studying gene expression, protein interactions, and drug screening. Luciferase also holds significant application prospects in medical diagnostics, cell imaging, and animal model research.

[0003] In some applications, such as biological imaging or in vivo imaging, traditional luciferases often lose activity or become ineffective at higher temperatures, which is not conducive to long-term in vivo monitoring of biological reactions, pathologies, and drug effects. In addition, in some molecular biology experiments or environmental testing, due to the high temperature requirements, the application of natural luciferases is limited due to their inherent thermal stability. With technological advances, scientists have obtained improved luciferases through genetic engineering and directed evolution. These improvements have optimized the performance of luciferases, significantly improving luminous efficiency, spectral characteristics, and stability. These new luciferases have further broadened their application range in different fields.

[0004] The small subunit luciferase derived from deep-sea shrimp was identified in 2000. After the discovery of natural luciferase, many researchers have modified the enzyme and developed enzymes with higher activity and wider application pH (6-9). However, the thermal stability 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 the present invention is to expand the application conditions of luciferase OLuc and make it suitable for large-scale industrial use, and to provide a luciferase OLuc mutant and its application.

[0006] The present invention achieves the above-mentioned purpose through the following technical solutions:

[0007] As a first aspect of the present invention, a luciferase OLuc mutant is provided. The luciferase OLuc mutant is obtained by modification based on the amino acid sequence of wild-type luciferase OLuc. 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, which encodes the luciferase OLuc mutant as described above, and the polynucleotide sequence encoding the luciferase OLuc mutant is shown in SEQ ID NO.3.

[0009] As a third aspect of the present invention, a recombinant plasmid is provided, which is 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, which comprises BL21 (DE3) cells containing any of the above-described recombinant plasmids or a genome in which the polynucleotide described above is integrated.

[0012] As a fifth aspect of the present invention, there is provided a method for preparing the luciferase OLuc mutant according to claim 1, characterized in that it comprises the following steps:

[0013] (1) synthesizing a gene sequence encoding the amino acid sequence of the luciferase OLuc mutant based on the amino acid sequence thereof, and then constructing the gene sequence into an expression vector to obtain a recombinant plasmid;

[0014] (2) Using an Escherichia coli prokaryotic expression system to express the recombinant plasmid obtained in step (1) to obtain an expression product, and purifying the expression product to obtain the luciferase OLuc mutant.

[0015] As a sixth aspect of the present invention, provided is a use of the above-described luciferase OLuc mutant in improving the yield or thermal stability of luciferase OLuc.

[0016] As a seventh aspect of the present invention, there is provided a use of the above-described luciferase OLuc mutant in molecular biology testing, food testing, environmental testing or medical testing.

[0017] The present invention has the following beneficial effects:

[0018] The present invention transforms the wild-type luciferase OLuc from deep-sea shrimp to obtain a luciferase OLuc mutant. The Tm value of the transformed luciferase OLuc mutant is 85.30°C, which has very high thermal stability. The luciferase OLuc mutant is 18.17°C higher than the Tm value of the reported luciferase NanoLuc, and the luciferase OLuc mutant still has a high luciferase activity, which can overcome the problem that the enzyme is unstable at high temperatures and limits its application, and provides an application scenario for its storage and better application in industrial production, giving it a wider range of application conditions. In addition, the yield of the luciferase OLuc mutant in Escherichia coli reached nearly 90 mg / L, which is 2.5 times higher than that of the luciferase NanoLuc. Its high yield also makes it more suitable for large-scale production, reducing the cost in subsequent application development. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The results of small-scale expression of the luciferase OLuc mutant and the control luciferase Nanoluc provided by the present invention;

[0020] Figure 2 The affinity purification results of the luciferase OLuc mutant and the control luciferase Nanoluc provided by the present invention;

[0021] Figure 3 Activity identification test results of the luciferase OLuc mutant and the control luciferase Nanoluc provided by the present invention;

[0022] Figure 4 These are the thermal stability test results of the luciferase OLuc mutant and the control luciferase Nanoluc provided by the present invention. DETAILED DESCRIPTION

[0023] The present application is described in further detail below in conjunction with the accompanying drawings. It is necessary to point out that the following specific implementation methods are only used to further illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Technicians in this field 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, the methods used in the present invention are conventional methods known to those skilled in the art. If specific conditions are not specified, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0026] 2. Methods

[0027] 2.1 Construction of recombinant luciferase plasmid

[0028] The gene sequences of the recombinant luciferase provided by the present 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. During recombinant expression, the signal peptide sequence at positions 1-27 of the N-terminus ("MAYSTLFIIALTAVVTQASSTQKSNLT") was truncated.

[0030] The wild-type luciferase OLuc sequence with the signal peptide sequence truncated was mutated to obtain a 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, the present invention also compared it with luciferase Nanoluc, which has a higher activity than wild-type luciferase OLuc (PDB ID 7SNS). The amino acid sequence of luciferase Nanoluc is shown in SEQ ID NO. 4. This enzyme is derived from wild-type luciferase OLuc and has a reported activity approximately 9-fold higher than wild-type luciferase OLuc (see Tomabechi, Yuri et al., "Crystal structure of nanoKAZ: The mutated 19 kDa component of Oplophorus luciferase catalyzing the bioluminescent reaction with coelenterazine.").

[0032] To facilitate protein purification, the tag 8His and the enzyme cleavage 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 synthesized luciferase genes were constructed on the expression vector pET-28a, and all recombinant plasmids were verified by sequencing to be completely consistent with the target sequence.

[0034] 2.2. Protein expression of recombinant luciferase

[0035] 2.2.1. Small-scale expression of recombinant luciferase

[0036] Using conventional molecular biological methods, the recombinant plasmids of the constructed luciferase OLuc mutant and the control luciferase Nanoluc were transformed into BL21 (DE3) E. coli competent cells in a clean bench and cultured at 37°C overnight. Monoclonal colonies cultured overnight were picked and transferred to 5 ml LB liquid medium and cultured at 37°C until the OD value of the bacterial solution reached 0. 600 When the pH value is 0.6-0.8, take a small amount of bacterial solution and fix it with loading buffer. Take a small amount of bacterial solution and add glycerol to freeze it at -80℃. Add 0.5mM IPTG to the remaining bacterial solution and induce it at 15℃ for 16 hours. Collect the bacteria and take the induced bacterial solution for SDS-PAGE detection.

[0037] See the results Figure 1 The luciferase OLuc mutant and the control luciferase Nanoluc were both significantly expressed in BL21 (DE3) Escherichia coli and had good protein solubility.

[0038] 2.2.2. Large-scale expression of recombinant luciferase

[0039] The strains with obvious expression were inoculated into 50 ml LB liquid medium and cultured at 37 °C overnight. The bacteria cultured overnight were inoculated into 1 L LB liquid medium at a ratio of 1:100 and cultured at 37 °C until the bacterial solution OD 600 When the pH value was 0.6-0.8, 0.5 mM IPTG was added, cultured overnight at 15°C, and the cells were collected by centrifugation at 5000 rpm.

[0040] 2.3 Protein Purification of Recombinant Luciferase

[0041] The collected bacteria were weighed, and the corresponding volume of lysis buffer (50mM Tris-HCl (pH 8.0), 500mM NaCl, 5% glycerol) was added in a ratio of 1:10. The bacteria were broken using a high-pressure homogenizer and the supernatant was collected by high-speed centrifugation at 16000rpm. All recombinant luciferases have 8His tags, and the protein was enriched and purified using a Ni Bestarose FF affinity chromatography column. The specific process is as follows: first, the Ni Bestarose FF affinity chromatography column was washed and balanced with lysis buffer for 10 column volumes, and then the lysis supernatant was loaded onto the Ni Bestarose FF affinity chromatography column, eluted with different gradients of imidazole solution, and the proteins eluted with different gradients of imidazole were collected for SDS-PAGE detection, and the protein concentration was measured with Nanodrop to calculate the protein yield.

[0042] Protein purification results are as follows Figure 2, obtaining a highly pure luciferase OLuc mutant and a control luciferase Nanoluc. Nanodrop assay results showed that 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. Comparing yields, the modified luciferase OLuc mutant achieved an approximately 2.5-fold increase in yield compared to the control luciferase Nanoluc.

[0043] 2.4. Activity test of recombinant luciferase

[0044] Luciferase binds to substrate and releases a detectable chemiluminescent signal. Higher luciferase activity indicates a greater number of photons produced per unit time by the catalytic reaction with the substrate, resulting in a greater intensity of the detected light signal. Enzyme activity is expressed as the intensity of the light signal absorbed per nanomole of protein.

[0045] The specific steps of the luciferase activity assay are as follows:

[0046] The substrate furimazine (GLPBiO, Catalog No.: PBI1939) was prepared as a 1 mM stock solution and aliquoted for later use. The luciferase activity assay buffer was 50 mM Tris-HCl (pH 8.0). The furimazine substrate concentration was diluted to 2 μM using the buffer, and the luciferase was diluted in a two-fold gradient from 100 nM, for a total of 12 dilutions. 10 μL of substrate was transferred to a 384-well plate and duplicated. 10 μL of the luciferase to be tested was transferred to the corresponding wells of the plate. Immediately centrifuge and shake to mix thoroughly. Fluorescence signals generated by the reaction were collected using a TECAN F200 microplate reader. Graph Pad Prism 9 analysis software was used for data analysis to determine the activity parameters of the protease to be tested. Figure 3 That is, the enzyme activity parameters of different luciferases were obtained by analyzing the Graph Pad Prism9 software.

[0047] The results of luciferase activity assay are as follows Figure 3 As shown, the activity of the modified luciferase OLuc mutant was measured to be 4314173 (RFU / nM), while the activity of the control luciferase Nanoluc was measured to be 8219667 (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 is approximately 9 times higher than that of wild-type OLuc. It can be seen that the luciferase OLuc mutant provided by the present invention still has good enzymatic activity.

[0048] 2.5 Thermal stability test of recombinant luciferase

[0049] The thermal stability test for recombinant luciferase uses the Protein Thermal Shift (ThermoFluor) technique. This technique exploits the structural characteristics of proteins, which contain hidden hydrophobic regions. When the temperature rises, the protein's structure opens, exposing the hydrophobic regions. The fluorescent dye SYPRO Orange binds to these regions and excites them to fluoresce. A melting curve is formed based on the intensity of the fluorescent signal. The temperature corresponding to the maximum value of the melting curve derivative is the melting point (Tm). The more stable the protein, the higher the measured Tm value.

[0050] The specific operation of the luciferase thermal stability assay is as follows:

[0051] 5 μg of luciferase was added to each well of a 96-well PCR plate, and 10× SYPRO Orange (Thermo Fisher Scientific, S6650) fluorescent dye was added to the corresponding wells. The 96-well PCR plate was placed in a qPCR instrument, and the instrument parameters were set. The temperature was raised from 20°C to 99°C in a gradient of 1°C per minute, and the protein melting curve was calculated.

[0052] The results are as follows Figure 4 As shown, the Tm value of the modified luciferase OLuc mutant is 85.30°C, while the Tm value of the control luciferase Nanoluc is 67.13°C. The modified luciferase OLuc mutant has significantly improved thermal stability, reaching above 85°C, an increase of 18.17°C 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, providing better storage and application scenarios in industrial production.

[0053] 3. Conclusion

[0054] The above results indicate that the luciferase OLuc mutant provided by the present invention has the characteristics of high protein yield and high thermal stability, and has good enzyme activity. It has broader application conditions and stronger practical application value, and is more suitable for large-scale production and industrial use.

[0055] The above-described embodiments merely illustrate several implementations of the present invention. 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 a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, and all such variations and improvements fall within the scope of protection of the present invention.

Claims

1. A luciferase OLuc mutant, characterized in that: The luciferase OLuc mutant is obtained by modification based on the amino acid sequence of the wild-type luciferase OLuc. 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 according to 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 according to claim 2 and capable of correspondingly translating and expressing the luciferase OLuc mutant according to 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 invention relates to a BL21 (DE3) cell containing the recombinant plasmid according to any one of claims 3 to 4 or a BL21 (DE3) cell in which the polynucleotide according to claim 2 is integrated into the genome.

6. A method for preparing a luciferase OLuc mutant according to claim 1, characterized in that: The following steps are involved: (1) Synthesizing a gene encoding the amino acid sequence of the luciferase OLuc mutant as shown in SEQ ID NO. 2, and then constructing the gene into an expression vector to obtain a recombinant plasmid; (2) The recombinant plasmid obtained in step (1) is expressed using an Escherichia coli prokaryotic expression system to obtain an expression product, and the expression product is purified to obtain the luciferase OLuc mutant.

7. Use of the luciferase OLuc mutant according to claim 1 in food testing or environmental testing.

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