A mutant gene, recombinant protein, plasmid, recombinant bacteria, preparation method and application

Abstract

This invention discloses a mutant gene, recombinant protein, plasmid, recombinant bacteria, preparation method, and application, belonging to the field of creatinine enzyme production technology. This invention will... Pseudomonas putida Codon optimization was performed on the CDS of the derived creatinine enzyme gene (cah) sequence (GenBank: AF170566.3) to identify the enzyme active site for translation of the optimized gene sequence. Mutation of the active site gene sequence enhanced the overall catalytic activity of creatinine enzyme after translation. The mutated gene sequence was inserted into the initial plasmid to construct a recombinant plasmid, which was then transformed into *E. coli* to prepare recombinant bacteria. The recombinant bacteria constructed in this invention significantly increased the yield and activity of creatinine enzyme during fermentation. High-purity recombinant creatinine enzyme can be obtained through a two-step purification process, which can help reduce costs and is of great significance for the development of precise renal function testing reagents.

Description

Technical Field

[0001] This invention relates to the field of creatinine enzyme production technology, specifically to a mutant gene, recombinant protein, plasmid, recombinant bacteria, preparation method, and application. Background Technology

[0002] Creatinine is formed from phosphocreatine in human muscles through a spontaneous and irreversible metabolic process. Under normal kidney function, the concentration of creatinine in the blood remains at a low level. However, in cases of kidney failure, creatinine accumulates in the serum. Therefore, serum creatinine levels are an important indicator of kidney function. The most widely used method for creatinine detection is the alkaline picric acid method, but it suffers from poor specificity and limited sensitivity. It is gradually being replaced by enzymatic methods (creatinine oxidase coupled with sarcosine oxidase). Creatine oxidase, creatinine oxidase, and creatine oxidase are three key enzymes. Creatine oxidase reversibly hydrolyzes creatinine to creatine, creatine oxidase catalyzes the hydrolysis of creatine to produce sarcosine and urea, and sarcosine is hydrolyzed by sarcosine oxidase to formaldehyde, glycine, and H₂O₂. Coupled with the Trinder reaction, the creatinine content in the sample is calculated by colorimetric determination. Enzymatic methods have the advantages of strong anti-interference ability, good specificity, wide linear range and high sensitivity. Most commercial kits use the creatinine enzyme coupled with sarcosine oxidase method and are increasingly used in clinical practice.

[0003] Creatinase is a key enzyme in enzymatic assay kits for creatinine detection. However, the production of this key enzyme in these kits currently relies heavily on imports. Domestically produced enzyme raw materials suffer from low fermentation yields and poor technical stability, resulting in consistently high production costs for creatinine assay kits. To reduce production and sales costs and alleviate the burden of testing for patients, it is crucial to develop highly active creatinine-producing strains to increase creatinine expression and activity, which is essential for promoting its widespread application in creatinine assay kits. Summary of the Invention

[0004] To address the aforementioned limitations of existing technologies, the present invention aims to provide a mutant gene, recombinant protein, plasmid, recombinant bacteria, preparation method, and application. This invention aims to utilize codon optimization and gene mutation techniques to synthesize a creatinine enzyme gene with optimized gene sequence, construct a recombinant expression vector, and thus obtain a highly efficient *E. coli* recombinant strain expressing recombinant creatinine enzyme. *E. coli* has a clear genetic metabolic network, simple culture conditions, is easy to operate in production, and has low fermentation costs, making it one of the most popular exogenous expression hosts currently available. Using... Pseudomonas putidaThe cah gene sequence was optimized based on the codon preference of Escherichia coli host bacteria. By identifying active sites and mutating the active site gene to enhance enzyme activity, soluble expression of the exogenous creatinine enzyme gene was achieved in Escherichia coli. Through research on the fermentation conditions and enzymatic properties of the recombinant bacteria, preliminary scale-up production under culture conditions was completed, aiming to provide theoretical basis and technical support for the industrial production of creatinine enzyme.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a mutant gene, wherein the mutant gene differs from the CDS of the cah sequence with GenBank number AF170566.3 in that a G→C mutation occurs at the 323rd base position, and the nucleotide sequence of the mutant gene is shown in SEQ ID NO:3.

[0007] In a second aspect, the present invention provides a recombinant protein expressed by the mutant gene of claim 1, which differs from the protein expressed by the cah sequence with GenBank number AF170566.3 in that the 108th amino acid is mutated from arginine to proline, and the amino acid sequence of the mutated protein is shown in SEQ ID NO:4.

[0008] A third aspect of the present invention provides a plasmid comprising the mutant gene.

[0009] In a fourth aspect, the present invention provides a recombinant bacterium, wherein the recombinant bacterium comprises the mutant gene or the plasmid.

[0010] A fifth aspect of the present invention provides a method for preparing the recombinant bacteria, comprising the following steps:

[0011] (1) The cah gene sequence was optimized, the gene active site was mutated, and then inserted into the initial plasmid to obtain the recombinant plasmid;

[0012] (2) The recombinant bacteria can be obtained by transforming the recombinant plasmid into Escherichia coli.

[0013] Furthermore, step (1) includes the following process:

[0014] Will Pseudomonas putidaThe source cah sequence was optimized, and the enzyme activity gene site was identified. Gene mutation was performed on the active site to obtain the optimized gene. The optimized gene sequence was amplified, and BamHI and XhoI restriction sites were introduced to obtain the amplified cah fragment. The initial plasmid and the amplified cah fragment were double-digested with BamHI and XhoI enzymes, respectively. After digestion and purification, the ligation vector product and the site-mutated cah fragment product were obtained, respectively. The vector product and the site-mutated cah fragment product were ligated to construct the recombinant plasmid.

[0015] Furthermore, the initial plasmid is pET-28a(+); the Escherichia coli is Escherichia coli BL21(DE3).

[0016] In some embodiments of the present invention, the cah sequence optimized by codon and gene site mutation is inserted between BamHI and XhoⅠ in plasmid pET-28a(+).

[0017] Furthermore, in step (2), the thermal shock method is used for conversion.

[0018] A sixth aspect of the present invention provides the use of the recombinant bacteria in the preparation of recombinant creatinine enzyme.

[0019] Furthermore, the recombinant bacteria were cultured in a fermentation medium, and the inducer isopropyl-β-D-thiogalactoside was added for induction culture. After the culture was completed, the bacterial cells were collected by centrifugation, and the recombinant creatinine enzyme was obtained by lysis and purification. The induction culture conditions were 25-35℃ and 0.1-1mM IPTG for 6-18h. The purification method was two-step purification: first purification with Ni-NTA, and then purification with ion exchange chromatography.

[0020] The beneficial effects of this invention are:

[0021] This invention introduces a codon-optimized and site-mutated creatinine enzyme gene into competent *E. coli* via a recombinant plasmid to construct a recombinant strain. The preparation method is simple. The *E. coli* recombinant strain provided by this invention can efficiently express recombinant creatinine enzyme, and high-purity recombinant creatinine enzyme can be obtained through two-step affinity purification, greatly improving the yield of creatinine enzyme and reducing the preparation cost of enzyme raw materials. The specific activity of the site-mutated group is increased by 130.3% compared to the non-mutated group, showing broad application prospects. Attached Figure Description

[0022] Figure 1 This is the molecular map of the pET-C recombinant plasmid constructed in Example 1 of the present invention.

[0023] Figure 2 This is a graph showing the expression efficiency of creatinine enzyme at different time points. Detailed Implementation

[0024] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0025] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0026] The method for preparing the recombinant Escherichia coli described in this invention is as follows: Pseudomonas putida The CDS of the source cah sequence (GenBank: AF170566.3) was codon optimized and mutated at gene sites, and then inserted into the initial plasmid to obtain a recombinant plasmid. The recombinant plasmid was then transformed into competent E. coli to obtain the final product.

[0027] In the recombinant Escherichia coli described in this invention, the initial plasmid includes, but is not limited to, plasmid pET-28a(+); in one embodiment of this invention, the initial plasmid is pET-28a(+), and the cah gene sequence is inserted between the BamHⅠ and XhoⅠ sites of plasmid pET-28a(+), and the resulting recombinant plasmid is denoted as pET-C (technically synthesized by GenScript).

[0028] The test materials used in the embodiments of the present invention, unless otherwise specified, are all conventional test materials in the art and can be purchased through commercial channels.

[0029] Example 1: Construction of recombinant bacteria

[0030] The specific implementation example demonstrates the construction of recombinant bacteria that efficiently express recombinant creatinine enzyme through the following experimental steps:

[0031] (1) Mutant genes and recombinant proteins

[0032] Codon optimization was performed on the cah sequence (GenBank: AF170566.3) to identify its enzyme activity catalytic site (GCACGGCGCGCGTCAGCT). Using site-directed mutagenesis, the CGG gene site at bases 322-324 of the CDS sequence was mutated to CCG, i.e., a G→C mutation occurred at base 323, yielding the mutant gene. The unmutated gene sequence is SEQ ID NO:1, and the mutated gene sequence is SEQ ID NO:3. At the amino acid level, this mutation changed amino acid 108 at the expression site from arginine to proline, thus obtaining the recombinant protein. The unmutated amino acid sequence is SEQ ID NO:2, and the mutated amino acid sequence is SEQ ID NO:4.

[0033] (2) Construction of recombinant plasmid pET-C

[0034] The cah gene sequence after sequence mutation and codon optimization was used as the sequence of the site-directed mutagenesis group; the cah gene sequence after the same codon optimization but without sequence mutation was used as the sequence of the unmutated group; the sequences of the site-directed mutagenesis group and the unmutated group were amplified separately.

[0035] The subsequent processing steps were identical for both the site-directed mutant and non-mutated sequences: BamHI and XhoI restriction sites were introduced to obtain the amplified cah fragment; the vector (pET-28a(+)) and the amplified cah fragment were double-digested with BamHI and XhoI, respectively, and then digested and purified to obtain the vector product and cah fragment product, respectively; the vector product and cah fragment product were ligated to construct the recombinant plasmid pET-C. The resulting recombinant plasmid pET-C is shown in the image below. Figure 1 As shown, the recombinant plasmid pET-C was added to Escherichia coli DH5α competent cells for heat transformation. After transformation, the bacterial strain was cultured, and colony PCR was performed to obtain the non-mutated recombinant plasmid and the site-directed mutagenic recombinant plasmid with correct sequencing results.

[0036] (3) Preparation of recombinant bacteria

[0037] Competent E. coli BL21(DE3) cells were removed from a -80°C freezer and placed on ice for 10 minutes. 2 μL of the unmutated recombinant plasmid and the site-mutated recombinant plasmid were added to 200 μL of chemically active *E. coli*, and the mixtures were allowed to stand on ice for 30 minutes. The mixtures were then heat-shocked in a 42°C water bath for 90 seconds, followed immediately by standing on ice for 2 minutes. 700 μL of LB liquid medium was added, and the mixture was shaken at 37°C for 1 hour. The resulting bacterial culture was centrifuged at 6000 × g at room temperature for 6 minutes, and the supernatant was removed. 100 μL of LB liquid medium was added, and the culture was plated on LB[Kan] plates and incubated overnight at 37°C in a shaker. After single colonies grew on the plates, the best-growing colonies were labeled. The results showed that recombinant bacteria expressing recombinant creatinine enzyme with either the site-mutated or unmutated cah sequence had been successfully constructed and were ready for use.

[0038] Example 2: Optimization of induced fermentation conditions for recombinant bacteria

[0039] Inoculate 3 mL of LB[Kan] culture medium containing the two recombinant bacteria prepared in Example 1. Take the equivalent of 1 mL and 1 unit OD 600The overnight bacterial solution was placed into 1.5 mL centrifuge tubes and immediately aliquoted. 2% of the overnight culture was inoculated into 10 mL LB [Kan] medium until the bacteria reached mesogaster growth (OD200). 600 =0.4~0.6). IPTG expression inducer was added to a specific concentration, and cultured at a specific temperature. Different time points were used during the induction period. The equivalent of 1 mL and 1 unit OD were added... 600 The *E. coli* culture was transferred to a 1.5 mL centrifuge tube. The tube was centrifuged at 8000 rpm for 2 minutes to remove the culture supernatant. The bacterial culture was resuspended in 50 μL of 1×SDS-PAGE Sample Loading Buffer and incubated at 95°C for 5 minutes. The culture was then centrifuged at 10000 rpm for 2 minutes and stored at -20°C. All prepared samples were loaded onto a gel. The sample to be tested was reheated to 95°C, and 10 μL of the suspension was loaded onto a 15% SDS-polyacrylamide gel.

[0040] Following the above procedure, 0.6 mM IPTG was used to induce creatinine enzyme expression. Samples were taken at 2, 4, 8 and 12 h of culture and then processed by SDS-PAGE.

[0041] Figure 2 The results showed that the optimal induction conditions for E. coli at 30°C and a concentration of 0.6 mM IPTG were 8 hours, resulting in the highest production efficiency. Therefore, the subsequent induction conditions can be optimized to 0.6 mM IPTG and an induction time of 8 hours.

[0042] Example 3: Recombinant bacteria induce expression of creatinine enzyme

[0043] Two recombinant bacteria prepared in Example 1 were used. Those containing the unmutated cah sequence were designated as the unmutated group, and those containing the mutated cah sequence were designated as the site-directed mutagenesis group. Expression was performed under the following induction conditions: 30°C, 0.6 mM IPTG for 8 h. After induction, *E. coli* cells were collected by centrifugation, and the wet weight of the bacterial precipitate was weighed using a balance. The precipitate was frozen overnight at -80°C, and then the *E. coli* cells were lysed. The particles were resuspended in binding / washing buffer. The circulating cooler was turned on, and when the temperature dropped below 4°C, the *E. coli* cells were pulverized using an ultrasonic bacterial lysis device until the liquid became clearly clear. The pulverized bacterial liquid was centrifuged at 10000 × g for 20 minutes at 4°C. The precipitate was insoluble and discarded; the supernatant was soluble, i.e., the crude enzyme solution containing creatinine enzyme. Creatinine enzyme was purified by eluting with a high concentration of imidazole (NI-NTA affinity chromatography).

[0044] To further improve enzyme purity, the product creatinine enzyme was purified in two steps. Specifically, the product was first purified using NI-NTA affinity chromatography, followed by further purification and desalting of the creatinine enzyme using ion exchange chromatography. After these two purification steps, the purity of the creatinine enzyme was increased to over 95.0%.

[0045] The activity of the obtained creatinine enzyme was tested. Enzyme activity units are defined as the amount of enzyme required to catalyze the reaction to produce 1 μmol of creatine per minute; specific activity refers to the enzyme activity per milligram of enzyme protein. The enzyme activity test results were as follows: After two-step chromatographic purification, the specific activity of the obtained creatinine enzyme was 162.74 U / mg in the non-mutated group and 212.03 U / mg in the site-directed mutagenesis group. The specific activity of the site-directed mutagenesis group was 130.3% higher than that of the non-mutated group.

[0046] In summary, this invention constructs a recombinant expression vector by optimizing the codon and mutating the creatinine enzyme gene at gene sites, thereby obtaining a highly efficient recombinant Escherichia coli strain expressing recombinant creatinine enzyme, which greatly increases the yield and activity of creatinine enzyme during fermentation.

[0047] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A mutant gene encoding creatinine enzyme, characterized in that, The mutated gene differs from the CDS of the cah sequence with GenBank number AF170566.3 only in that a G→C mutation occurs at the 323rd base position. The nucleotide sequence of the mutated gene is shown in SEQ ID NO:

3.

2. A recombinant protein, characterized in that, The recombinant protein is expressed by the mutant gene described in claim 1, and differs from the protein expressed by the cah sequence with GenBank number AF170566.3 only in that the amino acid at position 108 is mutated from arginine to proline, and the amino acid sequence of the mutated protein is shown in SEQ ID NO:

4.

3. A plasmid, characterized in that, The plasmid includes the mutant gene described in claim 1.

4. A recombinant bacterium, characterized in that, The recombinant bacteria include the mutant gene of claim 1 or the plasmid of claim 3.

5. The method for preparing the recombinant bacteria according to claim 4, characterized in that, Includes the following steps: (1) The cah gene sequence was optimized, the gene active site was mutated, and then inserted into the initial plasmid to obtain the recombinant plasmid; (2) The recombinant bacteria can be obtained by transforming the recombinant plasmid into Escherichia coli.

6. The method for preparing recombinant bacteria according to claim 5, characterized in that, Step (1) includes the following process: Will Pseudomonas putida The source cah sequence was optimized, and the enzyme activity gene site was identified. Gene mutation was performed on the active site to obtain the optimized gene. The optimized gene sequence was amplified, and BamHI and XhoI restriction sites were introduced to obtain the amplified cah fragment. The initial plasmid and the amplified cah fragment were double-digested with BamHI and XhoI enzymes, respectively. After digestion and purification, the ligation vector product and the site-mutated cah fragment product were obtained, respectively. The vector product and the site-mutated cah fragment product were ligated to construct the recombinant plasmid.

7. The method for preparing recombinant bacteria according to claim 6, characterized in that, The initial plasmid was pET-28a(+); the Escherichia coli was Escherichia coli BL21(DE3).

8. The method for preparing recombinant bacteria according to claim 5, characterized in that, In step (2), the thermal shock method is used for conversion.

9. The use of the recombinant bacteria according to claim 4 in the preparation of recombinant creatinine enzyme.

10. The application according to claim 9, characterized in that, The recombinant bacteria were cultured in fermentation medium, and the inducing agent isopropyl-β-D-thiogalactoside was added for induction culture. After the culture was completed, the bacterial cells were collected by centrifugation, and the recombinant creatinine enzyme was obtained by lysis and purification. The induction culture conditions were 25-35℃ and 0.1-1mM IPTG for 6-18h. The purification method was two-step purification: first purification with Ni-NTA, and then purification with ion exchange chromatography.

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