Luciferase OLuc mutant and application thereof

Abstract

The invention discloses a luciferase OLuc mutant and application thereof, and relates to the technical field of bioengineering, the luciferase OLuc mutant is obtained by modification on the basis of an amino acid sequence of wild-type luciferase (OLuc) from deep sea shrimps, and the amino acid sequence of the luciferase OLuc mutant is as shown in SEQ ID NO.2. Compared with reported NanoLuc modified on the basis of OLuc, the OLuc mutant has the characteristic of higher thermal stability, the Tm value of the OLuc mutant reaches 85 DEG C or above and is 18 DEG C or above higher than that of NanoLuc, the problem that the application of the OLuc mutant is limited due to the fact that enzyme is unstable at high temperature can be solved, an application scene is provided for storage of the OLuc mutant and better application of the OLuc mutant in industrial production, and the OLuc mutant has wider application conditions. In addition, the method is more suitable for large-scale production due to high yield, and the cost in subsequent application and development is reduced.

Description

Technical Field

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

[0002] Luciferase is a class of enzymes that can catalyze the luminescence of luciferin. It is widely present in various organisms, especially luminous organisms (such as fireflies, deep-sea organisms, etc.). The most famous representative of this enzyme is the luciferase from fireflies, which produces visible light (fluorescence) under specific conditions by catalyzing the reaction of luciferin with oxygen, ATP and divalent magnesium ions. In this process, the light emitted usually has a longer wavelength (such as blue light or green light), and the intensity of the light is closely related to the concentration of the reactants. Therefore, the light signal generated by the luciferase catalytic reaction has the characteristics of high sensitivity, low background noise and quantification, which makes it an ideal choice for molecular probes and sensors, and has been widely used in cell and molecular biology research. Luciferase reporter gene technology has become an important tool for studying gene expression, protein interaction, drug screening and other fields. In addition, luciferase also has important application prospects in medical diagnosis, cell imaging, animal model research and other aspects.

[0003] In some applications, such as biological imaging or in vivo imaging, traditional luciferases usually lose activity or become ineffective at higher temperatures, which is not conducive to long-term monitoring of biological reactions, lesions, drug effects, etc. in the body; 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 poor thermal stability. With the advancement of technology, scientists have obtained improved luciferases through genetic engineering and directed evolution. These improvements have made luciferases more optimized in performance, with significant improvements in 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 an enzyme with higher activity and a wider range of 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 the 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, wherein the polynucleotide 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, wherein the recombinant plasmid 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 a BL21 (DE3) cell containing any of the above-mentioned recombinant plasmids or a polynucleotide as described above integrated into its genome.

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

[0013] (1) synthesizing a gene sequence capable of encoding the amino acid sequence of the luciferase OLuc mutant according to the amino acid sequence thereof, and then constructing the gene sequence on 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, there is provided a use of the above-mentioned 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 luciferase OLuc mutant as described above in molecular biology detection, food detection, environmental detection or medical detection.

[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 luciferase OLuc mutant obtained by transformation 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, so that it has 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 small-scale expression results 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 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 further described in detail below in conjunction with the accompanying drawings. It is necessary to point out here 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. Technical personnel 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 no specific conditions are 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. The signal peptide sequence at positions 1-27 of the N-terminus ("MAYSTLFIIALTAVVTQASSTQKSNLT") was truncated during recombinant expression.

[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] In order to better compare the performance of the modified luciferase, the present invention is also compared with the luciferase Nanoluc with a protein structure database PDB ID number of 7SNS, which has a higher activity than the wild-type luciferase OLuc. The amino acid sequence of luciferase Nanoluc is shown in SEQ ID NO. 4. The enzyme is obtained by modification based on the wild-type luciferase OLuc, and its reported activity is about 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 (the amino acid sequence of 8His is "HHHHHHHH", and the amino acid sequence of 3C is "LEVLFQGP") were added to the N-terminus of the sequences shown in SEQ ID NO.2 and SEQ ID NO.4.

[0033] All the synthesized luciferase genes were constructed on the expression vector pET-28a, and all the 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 above-constructed luciferase OLuc mutant and the control luciferase Nanoluc were transformed into BL21 (DE3) Escherichia coli competent cells in a clean bench and cultured at 37°C overnight. The monoclonal colonies cultured overnight were picked and placed in 5 ml LB liquid culture medium and cultured at 37°C until the OD value of the bacterial solution reached 0. 600 When the p-value is 0.6-0.8, take a small amount of bacterial solution and fix it with loading buffer, and take a small amount of bacterial solution and add glycerol to freeze to -80℃, add 0.5mM IPTG to the remaining bacterial solution, induce at 15℃ for 16 hours, collect the bacteria and take the induced bacterial solution for SDS-PAGE detection.

[0037] 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 bacteria 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: 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 determined with Nanodrop to calculate the protein yield.

[0042] Protein purification results Figure 2, and obtained luciferase OLuc mutants and control luciferase Nanoluc with better purity. According to the Nanodrop measurement results, the protein yield of the modified luciferase OLuc mutant is about 90mg / L, and the protein yield of the control luciferase Nanoluc is about 35mg / L. Comparing the yields, it can be seen that the yield of the modified luciferase OLuc mutant is about 2.5 times higher than that of the control luciferase Nanoluc.

[0043] 2.4. Activity test of recombinant luciferase

[0044] Luciferase binds to substrate and releases chemiluminescent signals that can be detected. The higher the luciferase activity, the more photons are generated per unit time by catalyzing the substrate reaction, and the greater the intensity of the detected light signal. The enzyme activity parameter is expressed as the intensity of the light signal absorbed per nanomole of protein.

[0045] The specific operation of the luciferase activity assay is as follows:

[0046] The substrate Furimazine (GLPBiO, catalog number: PBI1939) was prepared into a 1mM stock solution and aliquoted for use. The luciferase activity assay buffer was 50mM Tris-HCl (pH 8.0). The substrate Furimazine concentration was diluted to 2μM with a buffer, and the luciferase was diluted from 100nM in a 2-fold gradient, with a total of 12 dilutions. 10μL of substrate was transferred to a 384-well plate and two replicate wells were set up. 10μL of the luciferase to be tested was transferred to the corresponding well plate, immediately centrifuged and oscillated to mix, and the fluorescence signal value generated by the reaction was collected using a TECANF200 microplate reader. Graph Pad Prism9 analysis software was used for data analysis, and the enzyme activity parameters of the protease to be tested were finally obtained. 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), and the activity of the control luciferase Nanoluc was measured to be 8219667 (RFU / nM). The activity of the modified luciferase mutant was about 1.9 times lower than that of the control luciferase Nanoluc, but according to previous reports, the activity of NanoLuc was about 9 times higher than that of the wild-type OLuc, which shows that the luciferase OLuc mutant provided by the present invention still has good enzyme activity.

[0048] 2.5. Thermal stability test of recombinant luciferase

[0049] The detection technology for the thermal stability test of recombinant luciferase uses protein thermal melting technology (Protein Thermal Shift, ThermoFluor). The detection is based on the structural characteristics of the protein. The protein has a hydrophobic region structure and is hidden inside. When the temperature rises, the structure of the protein will be opened, exposing the hydrophobic region. The fluorescent dye SYPRO Orange can bind to this region and be excited to fluoresce. A melting curve is formed according to the change in the intensity of the fluorescent signal. The temperature corresponding to the maximum value of the derivative of the melting curve is the melting point temperature (Tm). The more stable the protein, the larger 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 increased from 20°C to 99°C in a gradient of 1°C per minute, and the dissolution curve of the protein was calculated.

[0052] The results are as follows Figure 4 As shown, the Tm value of the modified luciferase OLuc mutant is 85.30℃, and the Tm value of the control luciferase Nanoluc is 67.13℃. The thermal stability of the modified luciferase OLuc mutant is significantly improved, which can be as high as 85℃ or above, and its value is 18.17℃ higher than that of Nanoluc. The modified luciferase OLuc mutant has high thermal stability, which can overcome the problem that the enzyme is unstable at high temperature and limits its application, and provides a better application scenario for its storage and application in industrial production.

[0053] 3. Conclusion

[0054] The above indicates 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, has broader application conditions and stronger practical application value, and is more suitable for large-scale production and industrial use.

[0055] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope 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 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 claimed in claim 2 and capable of correspondingly translating and expressing the luciferase OLuc mutant as claimed in claim 1.

4. The recombinant plasmid according to claim 3, characterized in that The expression vector is pET-28a.

5. An expression system of 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 sequence capable of encoding the amino acid sequence of the luciferase OLuc mutant as shown in SEQ ID NO.2, and then constructing the gene sequence on an expression vector to obtain a recombinant plasmid; (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.

7. A use of the luciferase OLuc mutant as claimed in claim 1 in improving the yield or thermal stability of luciferase OLuc.

8. A use of the luciferase OLuc mutant as claimed in claim 1 in molecular biology detection, food detection, environmental detection or medical detection.

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