Coding codon sequence of luciferase for optimized expression in escherichia coli

Abstract

The invention provides a coding codon sequence of luciferase which is optimally expressed in escherichia coli, and the soluble expression of the luciferase in the escherichia coli is promoted under the condition of not changing a protein amino acid sequence. The luciferase is obtained by constructing a recombinant vector, expressing and purifying the coding codon sequence, and the fluorescence intensity of the luciferase is obviously improved under the condition of the same protein concentration. The improvement is as follows: the codon sequence can more effectively promote synchronous dynamic folding along with the translation process by regulating and controlling the extension rate of the peptide chain, so that more luciferase can obtain a correct structure state, and a higher proportion of luminous protein molecules can be obtained.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular, to a codon sequence encoding luciferase for optimized expression in Escherichia coli. BACKGROUND

[0002] Luciferase is a class of oxidoreductases that can catalyze the oxidation of luciferin or its derivatives to emit light. It is widely present in organisms in nature, from deep-sea luminous jellyfish to fireflies, from bacteria to fungi. Luciferases of different origins have unique molecular structures and catalytic properties. In the process of catalytic reaction, luciferase uses oxygen to oxidize luciferin into oxidized luciferin, while converting chemical energy into light energy, releasing photons, producing biological fluorescence that can be seen by the naked eye or detected by instruments. This unique light-emitting mechanism does not require an external excitation light source, making detection more sensitive and less background interference. In the field of life sciences, luciferase has become a core tool for gene expression regulation, signal pathway research, and drug screening experiments. Scientists can monitor gene transcription and translation levels in real time and quantitatively by fusing luciferase genes with target genes to construct reporter gene systems, quickly assessing promoter activity or the impact of drugs on gene expression. In vivo imaging technology, luciferase-labeled cells or organisms can be used to track tumor metastasis, immune cell dynamics, and drug distribution, providing visual key data for disease mechanism research and innovative therapy development, greatly promoting the development of disciplines such as molecular biology, cell biology, and translational medicine.

[0003] Luciferase expression in Escherichia coli has both advantages and disadvantages, and also faces some technical challenges. Escherichia coli has the characteristics of rapid growth and low cultivation cost, can achieve large-scale expansion of luciferase in a short time, and improve production efficiency; its genetic background is clear, and gene manipulation technology is mature, making it easy to modify and optimize luciferase genes through genetic engineering; and the culture conditions of Escherichia coli are simple, easy to mass industrial production, which can reduce the preparation cost of luciferase.

[0004] However, Escherichia coli is a prokaryote, lacking the protein folding and modification system unique to eukaryotes, which may lead to incorrect folding of luciferase, affecting its activity and stability; in addition, Escherichia coli produces endotoxins during growth, which are difficult to completely remove during luciferase extraction, and may interfere with subsequent experiments and applications; at the same time, luciferase expressed by Escherichia coli sometimes forms inclusion bodies, which need a complex renaturation process to restore activity, increasing the difficulty and cost of production.

[0005] The main difficulties currently faced include how to optimize the expression conditions to improve the yield of soluble luciferase, how to efficiently remove endotoxins to ensure the quality and safety of luciferase products, and how to further improve genetic engineering techniques to enable E. coli to more efficiently express luciferase with natural activity to meet the needs of life science research and industrial applications. SUMMARY

[0006] The present application aims to provide a codon sequence for luciferase optimized for expression in E. coli, to address the technical problems of high production difficulty and high cost of luciferase expressed in E. coli.

[0007] To achieve the above-mentioned purpose, the present application provides a codon sequence for luciferase optimized for expression in E. coli, comprising any one of the following: (1) a DNA sequence as shown in SEQ ID NO. 1; (2) obtained by replacing the synonymous codons at the sites in Table 1 based on SEQ ID NO. 1; (3) an RNA sequence as shown in SEQ ID NO. 2; (4) obtained by replacing the synonymous codons at the sites in Table 2 based on SEQ ID NO. 2; Table 1 Table 2 To achieve the synergistic optimization of the expression efficiency of the target gene and the folding quality of the protein, the present application provides a list of synonymous codon replacement sites (Table 1 and Table 2) that can be implemented in the luciferase coding sequence, which lists the codon sites and their optional synonymous codon replacement combinations without changing the amino acid sequence of the target protein. The selection of the replacement sites is based on the codon usage bias in E. coli, the requirement for translation rate regulation, and the calculation results of the structural information-driven deep learning model (patent application 2025107176722, codon sequence design method and device based on large multi-modal model) constructed by the applicant of the present application.

[0008] The current codon of each numbered site and the set of synonymous codons that it can be replaced with are explicitly labeled in Table 1 and Table 2. For example, in Table 1, the "GGC" at the 10th site of SEQ ID NO. 1 can be replaced with "GGT", "GGA" or "GGG" to modulate the local translation rate and assist protein folding. Similarly, multiple "CCG", "GGT", "GGC" codons in SEQ ID NO. 1 can also be replaced with other synonymous codons to construct different optimized versions as needed.

[0009] The above-mentioned codon replacement scheme is not only applicable to the design of site-directed mutation at the DNA level, but also can be applied to mRNA construction and optimization of in vitro transcription expression system. The RNA sequence shown in SEQ ID NO. 2 is used to support biological synthesis pathways such as in vitro transcription, mRNA vaccine expression, or other expression systems that do not rely on DNA.

[0010] Preferably, the amino acid sequence of the luciferase is shown in SEQ ID NO. 3.

[0011] The present application also provides a recombinant expression vector containing the aforementioned coding codon sequence.

[0012] Preferably, the recombinant expression vector is obtained by connecting the aforementioned coding codon sequence to a pET28a vector.

[0013] The present application also provides a recombinant E. coli obtained by transforming the aforementioned recombinant expression vector.

[0014] The present application also provides a method for preparing luciferase, the specific steps of which are as follows: S1. Constructing a recombinant expression vector containing the aforementioned coding codon sequence; S2. Transforming the recombinant expression vector into E. coli to obtain a recombinant E. coli containing the recombinant expression vector; S3. Inducing expression; S4. Purification.

[0015] Preferably, in step S1, the recombinant expression vector is obtained by connecting the aforementioned coding codon sequence to a pET28a vector.

[0016] Preferably, the specific method of step S2 is to first transform the recombinant expression vector into competent BL21(DE3), and then perform primary culture and expansion culture.

[0017] Further preferably, the transformation operation is as follows: 4 μg of the freeze-dried recombinant expression vector is dissolved in 100 μL of sterile water to obtain a plasmid solution; 2 μL of the plasmid solution is added to 50 μL of competent BL21 (DE3), and is placed on ice for 20 minutes; 42℃ heat shock for 90 seconds, and then cooling on ice for 2 minutes; in the clean bench, 500 μL of LB medium is added to the competent cells, and is recovered at 37℃ for 40 minutes.

[0018] Further preferably, the initial culture method is as follows: 10 μL of kanamycin (concentration 50 mg / mL) is added to 5 mL of LB medium, and then 100 μL of the recovered competent cells are added, and are cultured overnight at 37℃ on a shaker.

[0019] Further preferably, the obtained bacterial solution of the initial culture is stored according to the following conditions: 800 μL of the bacterial solution is stored in 20% glycerol, and is stored in a -20℃ refrigerator.

[0020] Further preferably, the expansion culture method is as follows: 300 μL of kanamycin (concentration 50 mg / mL) and 3 mL of the bacterial solution are sequentially added to 250 mL of LB medium, and are cultured at 37℃ and 220 rpm on a shaker for 4 hours.

[0021] Further preferably, the formula of the LB medium is as follows: 10 g / L of tryptone, 5 g / L of yeast extract, and 10 g / L of sodium chloride are dissolved in distilled water, and are autoclaved at 121℃ for 20 minutes.

[0022] Preferably, in step S3, isopropyl-beta-D-thiogalactopyranoside is used for inducing expression.

[0023] Preferably, the specific method of step S4 is as follows: the bacterial cells are collected by centrifugation, are resuspended, are broken by ultrasonic, are centrifuged to obtain the supernatant, and are purified by affinity chromatography.

[0024] Further preferably, the specific method of the affinity chromatography purification is as follows: the supernatant is purified by Ni-NTA affinity chromatography, the impurities are first washed with PBS containing 20 mM imidazole, and then the target protein is eluted with PBS containing 250 mM imidazole and PBS containing 500 mM imidazole.

[0025] Further preferably, the PBS containing imidazole is based on the following formula: 8 g / L of NaCl, 0.2 g / L of KCl, 1.44 g / L of Na2HPO4, and 0.24 g / L of KH2PO4, and then hydrochloric acid is used to adjust the pH to 7.4, and then the corresponding concentration of imidazole is added.

[0026] The present application has the following beneficial effects: The codon sequence encoding the luciferase provided by the present application promotes the soluble expression of luciferase in E. coli without changing the amino acid sequence of the protein.

[0027] The codon sequence encoding the luciferase provided by the present application promotes the soluble expression of luciferase in E. coli without changing the amino acid sequence of the protein.

[0028] The luciferase obtained by constructing a recombinant vector, expression and purification of the codon sequence encoding the luciferase of the present application has a significantly improved fluorescence intensity under the same protein concentration. This improvement is due to the regulation of the peptide chain elongation rate by the gene sequence of the present application, which can more effectively promote the synchronous dynamic folding accompanying the translation process, so that more luciferase has the correct structure state, and thus has a higher proportion of protein molecules that can emit light.

[0029] In addition to the purposes, features and advantages described above, the present application has other purposes, features and advantages. The present application will be further described in detail below with reference to the drawings. BRIEF DESCRIPTION OF DRAWINGS

[0030] The accompanying drawings, which form a part of this application, are included to provide a further understanding of the application, illustrate the preferred embodiments of the application, and assist in the explanation of the application. In the drawings: Figure 1 is the SDS-PAGE detection result, the left is the present application, and the right is the comparison sequence. DETAILED DESCRIPTION

[0031] The embodiments of the present application will be described in detail below with reference to the drawings, but the present application can be implemented in various different ways as limited and covered by the claims.

[0032] The main materials involved in the embodiments of the present application are as follows: The host bacteria E. coli BL21 (DE3) was purchased from GenScript Biotech; Phosphate buffered saline (PBS): prepared according to NaCl 8g / L, KCl 0.2g / L, Na2HPO41.44g / L, KH2PO40.24g / L, pH=7.4, premixed powder purchased from GenScript Biotech; Imidazole-containing PBS is based on NaCl 8 g / L, KCl 0.2 g / L, Na2HPO4 1.44 g / L, KH2PO4 0.24 g / L, and then adding the corresponding concentration of imidazole; The LB medium formula is: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, dissolved with distilled water, and autoclaved at 121°C for 20 minutes; Bright-Luc™ Firefly Luciferase Reporter Gene Detection Kit, TransGen Biotech Company.

[0033] Examples A method for preparing luciferase, the specific steps are as follows: S1. Constructing a recombinant expression vector containing a coding codon sequence The nucleotide sequence of the coding codon sequence is shown in SEQ ID NO. 1, SEQ ID NO. 2.

[0034] The comparison sequence is shown in SEQ ID NO. 4.

[0035] The amino acid sequence of luciferase is shown in SEQ ID NO. 3.

[0036] The coding codon sequence and the comparison sequence are respectively connected to the pET28a vector (purchased from GenScript Biotech) to obtain the corresponding recombinant expression vector.

[0037] Synthesized by GenScript Company, and the company feedback freeze-dried plasmid.

[0038] S2. Transforming the recombinant expression vector into E. coli to obtain recombinant E. coli containing the recombinant expression vector Plasmid dissolution: dissolve 4 μg of freeze-dried plasmid with 100 μL of sterile water.

[0039] Plasmid transformation: Take 2 μL of plasmid solution and add it to 50 μL of competent BL21 (DE3), and let it stand on ice for 20 min; 42°C heat shock for 90s, then cool on ice for 2 min; In the clean bench, add 500 μL of LB medium to the competent cells, and recover at 37°C for 40 min.

[0040] Bacterial liquid culture and preservation: Primary culture: take 10 μL of kanamycin (concentration 50 mg / mL) and add it to 5 mL of LB medium, then add 100 μL of recovered competent cells, and culture overnight at 37°C on a shaker.

[0041] Bacterial liquid preservation: Take 800 μL of bacterial liquid in logarithmic growth phase or stable phase, and mix with 200 μL of 80% (v / v) pre-prepared and sterilized glycerol water solution in a sterile cryogenic tube. The final volume concentration of glycerol after mixing is 20%. After mixing, store in a -20°C refrigerator for short-term preservation.

[0042] Expansion culture: Add 300 μL of kanamycin (concentration 50 mg / mL), 3 mL of bacterial liquid to 250 mL of LB medium (stored in a 500 mL conical flask) in sequence, and culture at 37°C, 220 rpm on a shaking table for 4 hours.

[0043] S3. Inducing expression Add 150 μL of isopropyl-β-D-thiogalactopyranoside (concentration 1 M) to 250 mL of LB medium, and culture at 16°C, 220 rpm overnight (14-16 hours).

[0044] S4. Purification OD600 determination: After connecting the ultraviolet spectrophotometer, set the OD600, and take 2 mL of pure LB culture in a cuvette, and place it in the instrument to zero. Take out the cuvette, and wash it with ddH2O, 70% alcohol, and ddH2O in sequence, and dry it. Take 2 mL of bacterial liquid to the cuvette, and record the value.

[0045] Centrifugal collection of bacterial cells: Take a fixed volume of bacterial liquid in a 250 mL centrifuge bottle, use a platform drop centrifuge and rotor, centrifuge at 12000 rpm for 5 min, and remove the supernatant.

[0046] Resuspension of bacterial cells: Resuspend the precipitate with 5 mL of PBS, and transfer it to a 50 mL centrifuge tube. Wash the 250 mL centrifuge tube with PBS 3 times, and finally resuspend the liquid to a volume of 11 mL.

[0047] Ultrasonic disruption and centrifugation: Set the ultrasonic probe power to 100%, and use a mode of 7s working and 3s resting to ultrasonically disrupt for 10 min, and store on ice after ultrasonication. Use a centrifuge to centrifuge at 12000 rpm for 40 min, and use a dropper to take 10 mL of supernatant (try to avoid the part close to the precipitate) in a small beaker, and store on ice. Completely remove the supernatant, resuspend the precipitate with 10 mL of PBS. This PBS is also prepared according to NaCl 8 g / L, KCl 0.2 g / L, Na2HPO4 1.44 g / L, KH2PO4 0.24 g / L, and adjust the pH to 7.4 with HCl, and autoclave at 121°C for 20 min.

[0048] Protein purification and detection: Affinity chromatography purification: The supernatant was purified by Ni-NTA affinity chromatography, and the impurities were washed with PBS containing 20 mM imidazole, and then the target protein was eluted with PBS containing 250 mM imidazole and PBS containing 500 mM imidazole.

[0049] Protein quantification: The total amount of eluted protein at each concentration was determined by nanodrop, and the total protein amount was the sum of the protein eluted by 250 mM and 500 mM imidazole.

[0050] SDS-PAGE detection: 200 μL before induction and 100 μL after induction were centrifuged at 12000 rpm for 2 min, the supernatant was removed, and 40 μL PBS was added for resuspension. The precipitate and post-system sample were diluted 10 times with PBS, and 40 μL was taken. 10 μL of 5x SDS-PAGE loading buffer (250 mM Tris-HCl (pH 6.8), 10% (w / v) SDS, 50% (v / v) glycerol, 25% (v / v) β-mercaptoethanol, and 0.05% (w / v) bromophenol blue) was added, and SDS-PAGE electrophoresis detection was performed.

[0051] Test example The luciferase sample obtained by purification in the example was dissolved in a phosphate buffered saline (PBS) eluent containing 250 mM imidazole.

[0052] Detection working solution in the Bright-Luc™ Firefly Luciferase Reporter Gene Detection Kit produced by TransGen Biotech was added to the sample. After gentle mixing, the chemiluminescence value was immediately detected on a multifunctional enzyme label instrument.

[0053] After the luminescence signal of the reaction system reached the platform period, the SpectraMax® i3x multifunctional enzyme label instrument was used for detection. The detection wavelength was set to 570 nm, and the chemiluminescence intensity (Relative Light Units, RLU) was recorded. The luminescence intensity value was used to characterize the biological activity of the purified luciferase.

[0054] The comparison results are shown in Table 3, which show that, under the condition of almost the same protein concentration, the luciferase expressed by the coding codon sequence of the present application has higher luminescence intensity than the protein expressed by the comparison sequence, and the former is about 50% higher than the latter. Considering that the protein concentration is consistent, this result must be caused by the difference in protein folding. The coding sequence of the present application is more conducive to synchronous dynamic folding during protein expression process, and can more efficiently achieve the correct folding form, so that the obtained protein has a larger proportion of molecules in the functional correct structure state.

[0055] Table 3 The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A codon sequence encoding a luciferase optimized for expression in E. coli, characterized in that, The DNA sequence as shown in SEQ ID NO. 1; (2) obtained by replacing the synonymous codons at the sites in Table 1 based on SEQ ID NO. 1; The RNA sequence as shown in SEQ ID NO. 2; (4) obtained by replacing the synonymous codons at the sites in Table 2 based on SEQ ID NO. 2; Table 1 Table 2 2. A recombinant expression vector containing the coding codon sequence of claim 1. The recombinant expression vector is obtained by connecting the aforementioned coding codon sequence to a pET28a vector.

4. A recombinant E. coli obtained by transforming the recombinant expression vector of claim 2. The specific steps are as follows: S1. Constructing a recombinant expression vector containing the coding codon sequence of claim 1; S2. Transforming the recombinant expression vector into E. coli to obtain a recombinant E. coli containing the recombinant expression vector; 。 S3. Inducing expression; 3. The recombinant expression vector of claim 2, wherein, S4. Purification. In step S1, the recombinant expression vector is obtained by connecting the aforementioned coding codon sequence to a pET28a vector.

5. A method for producing luciferase, characterized by, The specific method of step S2 is: first, transforming the recombinant expression vector into competent BL21 (DE3), and then performing primary culture and scale-up culture. In step S3, isopropyl-beta-D-thiogalactopyranoside is used for inducing expression. The specific method of step S4 is: centrifuging to collect the bacterial cells, resuspending the bacterial cells, ultrasonic crushing, centrifuging to obtain the supernatant, and affinity chromatography purification. ​ ​ 6. The method for preparing luciferase according to claim 5, characterized in that, ​ 7. The method for preparing luciferase according to claim 5, characterized in that, ​ 8. The method for preparing luciferase according to claim 5, characterized in that, ​ 9. The method for preparing luciferase according to claim 5, characterized in that, ​

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