Preparation method of UrdA urocanic acid reductase
By expressing and purifying UrdA urocanate reductase in Rosetta (DE3) strain, the problem of inaccurate enzyme activity assessment in existing technologies has been solved, and the standardized preparation and application of urocanate reductase has been realized, supporting subsequent research and applications.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHONGSHAN HOSPITAL FUDAN UNIV
- Filing Date
- 2026-03-06
- Publication Date
- 2026-05-12
AI Technical Summary
Current research on uric acid reductase relies on expression by natural strains or complex microbial culture systems, resulting in poor reproducibility of experimental results. Furthermore, the lack of standardized enzyme products makes it difficult to conduct independent, accurate, and quantitative enzyme activity assessments, thus limiting the in-depth development of related research.
The UrdA gene with optimized codons was cloned into the pSHA-2 (His-tag) expression vector, transformed into Rosetta (DE3) strain, expressed by IPTG, and purified by Ni-Smart affinity chromatography. Combined with dialysis and ultrafiltration concentration, high-purity and high-activity UrdA urocanate reductase was obtained.
This study enables reproducible, scalable, and stable preparation of uric acid reductase, provides standardized experimental materials, establishes a unified enzyme activity detection system, and supports basic and applied research related to uric acid reductase.
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Figure CN122012424A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedicine, specifically to a method for preparing UrdA urocanate reductase. Background Technology
[0002] As the role of gut microbiota metabolism in metabolic diseases, cardiovascular diseases, and inflammation-related diseases becomes increasingly recognized, metabolic enzymes derived from gut microbes and their catalytic reaction processes have become a focus of research and application. Among them, urocanate reductase (UrdA), as a key enzyme in the histidine metabolic pathway, can catalyze the conversion of urocanate to imidazole propionic acid, and is an important node connecting gut microbial metabolic activities with the host's metabolic state.
[0003] Existing research indicates that imidazole propionate is elevated in various metabolic abnormalities, and its production is highly dependent on the expression and catalytic activity of UrdA in gut microbiota. Therefore, the enzymatic properties, catalytic mechanisms, activity regulation, and inhibition strategies of UrdA have become key areas of focus in basic research, drug screening, and functional intervention studies.
[0004] However, current research on uric acid reductase mainly relies on expression in natural bacterial strains or complex microbial culture systems. Since UrdA is mostly derived from anaerobic or facultative anaerobic microorganisms, its expression level is significantly affected by strain background, culture conditions, and environmental factors, leading to poor reproducibility of experimental results between different studies. Furthermore, natural bacterial systems often contain multiple metabolic enzymes and interfering factors, making it difficult to independently, accurately, and quantitatively assess the enzyme activity of UrdA.
[0005] More importantly, to date, there are no commercially available reagents or standardized enzyme products for uric acid reductase. Researchers cannot directly obtain purified UrdA protein for in vitro reaction system construction, inhibitor screening, kinetic parameter determination, or detection method establishment. This situation objectively limits the in-depth development of UrdA-related research and also restricts its further development in detection, intervention, and translational applications.
[0006] In the absence of commercially available UrdA enzymes, developing a reproducible, scalable, and stable method for constructing uric acid reductase has become an urgent need in the current technological field. Summary of the Invention
[0007] The purpose of this application is to provide a method for preparing UrdA urocanate reductase, in order to solve the problems of limited enzyme sources, unstable preparation process and lack of standardized reagents in the prior art, so as to apply it in subsequent in vivo and in vitro experiments such as enzyme catalytic reactions and inhibitor screening.
[0008] To achieve the above objectives, the specific technical solution adopted by the present invention is as follows:
[0009] In a first aspect, this invention provides a method for preparing UrdA urocanate reductase, comprising steps such as expression plasmid construction, expression testing, amplification and affinity purification, protein dialysis, and protein concentration. The concentrated UrdA urocanate reductase exhibits high purity and good activity. The specific steps of this method are as follows:
[0010] S1. The codon-optimized UrdA gene was cloned into the pSHA-2 (His-tag) expression vector to construct a recombinant expression plasmid; the sequence of the codon-optimized UrdA gene is shown in SEQ ID NO.2;
[0011] S2. The recombinant expression plasmid was transformed into Rosetta(DE3) strain, and UrdA protein was expressed by IPTG induction;
[0012] S3. After ultrasonic disruption of bacterial cells, the supernatant was collected and the protein was purified using Ni-Smart affinity chromatography;
[0013] S4. Dialyze the purified protein;
[0014] S5. The dialysate is concentrated by ultrafiltration to obtain high-purity, high-activity UrdA protein.
[0015] Preferably, in step S1, the expression vector has kanamycin resistance and its cloning site is BamHI-XhoI.
[0016] Preferably, in step S2, the method for transforming the recombinant expression plasmid is as follows: after mixing the recombinant expression plasmid with Rosetta (DE3) cells, incubate on ice, then heat shock in a 42°C water bath for 80-100 seconds, and place on ice for 4-6 minutes after heat shock.
[0017] Preferably, in step S2, the method for inducing UrdA protein expression by IPTG is as follows:
[0018] Add LB liquid medium to the mixture of heat-shocked recombinant expression plasmid and Rosetta (DE3) cells, and incubate at 37°C and 200-250 rpm for 45-60 minutes;
[0019] The culture was inoculated onto LB agar plates and incubated overnight at 37°C.
[0020] The cultured strain was inoculated into LB liquid medium and cultured at 37°C and 220 rpm until the bacterial OD600 reached 0.4-0.6. IPTG was added to the culture to a concentration of 0.2 mM, and the culture was continued at 16°C and 220 rpm for 16 h to induce the expression of the fusion protein.
[0021] Preferably, in step S3, the bacterial cells are resuspended in PBS, β-mercaptoethanol and PMSF are added, and the cells are sonicated in an ice bath for 3 seconds, paused for 3 seconds, and sonicated 90-110 times; then centrifuged and the supernatant is collected.
[0022] Preferably, in step S3, the eluent used for protein purification by Ni-Smart affinity chromatography has the following composition: 20 mM PB, 0.5 M NaCl, 100 mM Imidazole, pH 7.4.
[0023] Preferably, in step S4, the dialysis method involves adding the purified protein to a PBS solution containing glycerol at pH 7.4 and dialyzing overnight at 4°C.
[0024] Preferably, in step S5, ultrafiltration concentration is carried out at 4000-5000 rpm and 4°C under low-shear centrifugation conditions, which effectively avoids local over-concentration of protein and conformational damage, thereby obtaining high-purity and high-activity UrdA protein.
[0025] Secondly, the present invention provides UrdA urocanic acid reductase prepared using the above-described preparation method. The above-described preparation method can reproducibly, scale up, and stably prepare urocanic acid reductase, overcoming the difficulties of directly culturing anaerobic bacteria and extracting it from anaerobic bacteria in existing enzyme preparation methods. The present invention utilizes the above-described preparation method to obtain commercially available UrdA urocanic acid reductase.
[0026] Thirdly, this invention provides the application of UrdA urocanic acid reductase prepared by the above preparation method in basic research, including screening of UrdA urocanic acid reductase inhibitors, research on UrdA urocanic acid reductase catalytic reactions, and research on intestinal flora metabolism-related diseases.
[0027] This invention provides a purified UrdA protein with high purity and good stability. It can be used for applications such as constructing in vitro reaction systems related to UrdA reductase and screening inhibitors. It overcomes the problems in existing UrdA reductase research, which relies on natural strain expression or complex microbial culture systems. These problems include the significant influence of strain background, culture conditions and environmental factors on UrdA expression levels, resulting in poor reproducibility of experimental results between different studies. Furthermore, the presence of various metabolic enzymes and interfering factors in natural bacterial systems makes it difficult to independently, accurately and quantitatively assess the enzyme activity of UrdA.
[0028] The present invention has the following beneficial effects:
[0029] Currently, there is no reproducible, scalable, and stable method for constructing uric acid reductase. This invention enables the construction of UrdA enzymes with a clearly derived, structurally stable, and biologically active form. This not only provides standardized experimental materials for related basic research but also helps establish a unified enzyme activity detection system, evaluate the effectiveness of regulatory methods, and lay the foundation for subsequent application-oriented product development. This invention is of great significance for further research on the screening and clinical translation of UrdA uric acid reductase inhibitors. Attached Figure Description
[0030] Figure 1 pSHA-2 (His-tag) plasmid map.
[0031] Figure 2 SDS-PAGE results of UrdA urokanolate reductase protein expression induced at 37℃. In the figure, the samples in each lane are: M: Marker; Lane 1: Supernatant protein; Lane 2: Inclusion body protein.
[0032] Figure 3 SDS-PAGE results of UrdA urokanolate reductase protein expression assay at 16℃. In the figure, the samples in each lane are: M: Marker; Lane 1: Supernatant protein; Lane 2: Inclusion body protein.
[0033] Figure 4 Results of SDS-PAGE affinity purification of UrdA urocanate reductase protein. In the figure, the samples in each lane are: M: Marker; Lane 1: Supernatant protein; Lane 2: Flow-through; Lanes 3-4: Elution sample.
[0034] Figure 5 Results of UrdA urokanolate reductase protein concentration. In the figure, the samples in each lane are: M: Marker; Lane 1: Concentrated sample. Detailed Implementation
[0035] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. Unless otherwise specified, the reagents used in the following embodiments are all commercially available conventional reagents, and the experimental procedures involved are all conventional procedures in the art unless otherwise specified.
[0036] Example 1
[0037] This embodiment provides a method for preparing UrdA urocanate reductase, including steps such as expression plasmid construction, expression testing, scale-up expression and affinity purification, protein dialysis, and protein concentration. After concentration, high-purity, high-activity UrdA urocanate reductase is obtained. The specific steps are as follows:
[0038] 1) Expression plasmid construction
[0039] The pSHA-2 (His-tag) expression vector with kanamycin resistance was used. Specifically, the empty vector map of the kanamycin-resistant pSHA-2 (His-tag) expression vector is shown below. Figure 1 As shown, the cloning site is BamHI-XhoI. In the pSHA-2 (His-tag) expression vector, the BamHI site is located downstream of the promoter, and the XhoI site is located upstream of the C-terminal tag or stop codon, ensuring that the inserted fragment can only be connected in one direction, avoiding reverse insertion, frameshift mutations, and His-tag reading errors.
[0040] The expression strain was Rosetta(DE3), derived from Sigma-Aldrich, Rosetta(DE3) CompetentCells – Novagen (NO.71397).
[0041] The UrdA urokinase reductase protein sequence is: EYTYDVVIIGSGGAGFSAGLEAIAAGRSAVIIEKMPIIGGNSLISGAEMNVAGSWVQKNMGITDSKELFISDTLKGGDFKGDPEMVKTMVDNAVGAAEWLRDYVKVEFYPDQLFQFGGHSVKRALIPKGHTGAEVISKFSIKADEVGLPIHTNTKAEKLIQDQTGRIVGVEAAHNGKTITYHAKRGVVIATGGFSSNMEMRKKYNPELDERYGSTGHAGG TGDGIVMAEKIHAAAKNMGYIQSYPICSPTSGAIALIADSRFFGAVLINQKGERFVEELERRDVISHAILAQPGRYTYVLWNQDIENVAHTVEMHQGELKEFTKDGLMYKVDTLEEAA KVFNIPEDKLLSTIKDVNHYAATGKDEAFNHRSGLVDLSKGPYWILKATPSVHHTMGGLVVDTRTRVLDEQGKVIPGLFAAGEVTGLTHGTNRLGGNAYTDIIVYGRIAGQEAAK (SEQ ID NO.1).
[0042]
[0043] After codon optimization of the target gene, corresponding restriction endonuclease sites (BamHI and XhoI) were introduced at its 5′ and 3′ ends, respectively, and specific PCR primers were designed accordingly. Using the optimized target gene as a template, PCR amplification was performed using high-fidelity DNA polymerase. After the amplification reaction, the PCR products were analyzed by agarose gel electrophoresis to confirm the size and specificity of the amplified fragments, and the target band was subsequently purified by gel electrophoresis.
[0044] Meanwhile, the empty expression plasmid vector was double-digested with BamHI and XhoI to obtain a linearized vector. After the digestion reaction, the target band was separated by agarose gel electrophoresis, and the linearized vector was purified by gel extraction to remove incompletely digested plasmids and small molecule impurities.
[0045] The purified target gene insert was ligated into a linearized vector to obtain a recombinant plasmid. The ligation product was then transformed into competent Rosetta (DE3) E. coli cells and plated on LB agar containing the appropriate antibiotics for selection.
[0046] After overnight incubation, four single colonies were randomly selected for colony PCR identification to preliminarily screen for positive clones. Colonies with positive PCR results were further incubated overnight with shaking at 37 °C. Part of the culture was used to prepare glycerol strains and stored at low temperature, while the other part was used for plasmid extraction. Finally, the sequence and insertion direction of the target gene in the recombinant plasmid were confirmed by Sanger sequencing, yielding the recombinant plasmid.
[0047] 2) Expression test
[0048] Remove competent Rosetta (DE3) cells from the cryogenic freezer and place them on crushed ice for 5 minutes. Add 100 ng of recombinant plasmid DNA to the competent cells and mix gently. Incubate on ice for 30 minutes, then heat shock in a 42°C water bath for 90 seconds and place on ice for 5 minutes.
[0049] Add 900 μL of antibiotic-free LB medium at room temperature, incubate at 37°C and 220 rpm for 45-60 minutes, centrifuge at 4500 rpm for 2 minutes, carefully aspirate the supernatant, gently mix the remaining liquid with a pipette, transfer it to an LB agar plate, spread it evenly, and incubate the plate inverted at 37°C overnight.
[0050] A small-scale test was conducted to compare protein expression under two induction methods: Two 5ml tubes of LB medium were prepared and inoculated with the bacterial strains, then cultured at 37℃ and 220 rpm until the bacterial OD600 reached 0.4-0.6. IPTG was added to one tube of culture to a final concentration of 1.0mM, and the culture was further incubated at 37℃ and 220 rpm for 4 hours to induce fusion protein expression. The culture was then collected and analyzed by SDS-PAGE. IPTG was added to the other tube of culture to a final concentration of 0.2mM, and the culture was further incubated at 16℃ and 220 rpm for 16 hours (the protein expression rate is reduced at 16℃, and the amount of protein synthesized per unit time is correspondingly reduced, so a longer induction time is required to reach a certain expression level) to induce fusion protein expression. The culture was then collected and analyzed by SDS-PAGE.
[0051] The specific sampling and testing process is as follows: 900 μL of the induced culture was aspirated, and the bacterial cells were collected by centrifugation. The precipitate was resuspended in 900 μL of 1×PBS, followed by sonication to lyse the bacteria. The supernatant proteins and inclusion body proteins were separated by centrifugation at 12000 rpm for 5 min, and samples were prepared separately for SDS-PAGE analysis. The results are as follows: Figure 2-3 As shown, under induction at 37℃, the protein was mainly expressed in the protein inclusion bodies of the bacterial cell fragments, while under induction at 16℃, the protein was mainly expressed in the supernatant of the bacterial cell fragments, which is more suitable for further nickel column purification. Therefore, we chose to induce large-scale expression and purification at 16℃ for subsequent purposes.
[0052] 3) Magnified expression and affinity purification
[0053] Inoculate the strain into 400 ml of LB medium and incubate at 37°C and 220 rpm until the bacterial cell OD reaches zero. 600 To a final concentration of 0.2 mM, add IPTG to the culture and incubate at 16°C and 220 rpm for 16 h. Collect the bacterial cells by high-speed centrifugation. Resuspend the bacterial cells in 30 ml of 1×PBS, add 30 μL of β-mercaptoethanol and 300 μL of PMSF, and sonicate on ice for 100 cycles (3 seconds sonication followed by 3 seconds pause). Centrifuge at 12000 rpm at 4°C for 15 min, and transfer the supernatant protein to a new tube.
[0054] Ni-Smart affinity chromatography was used to purify the supernatant protein. The buffer preparation method is as follows: Equilibration buffer: 20 mM PB, 0.5 M NaCl, pH 7.4; Washing buffer: 20 mM PB, 0.5 M NaCl, 10 mM Imidazole, pH 7.4; Elution buffer: 20 mM PB, 0.5 M NaCl, 100 mM Imidazole, pH 7.4 (3.0 ml packing material, loading ~8 mg / ml His-tag protein, flow rate about 1 ml / min, room temperature column chromatography).
[0055] Wash the column once with 15 ml deionized water; equilibrate once with 5 ml equilibration buffer; load the sample, repeat once, collect the permeate and retain 20 μl for electrophoresis; wash the column once with 15 ml washing buffer, collect the wash and retain 20 μl for electrophoresis; elute the protein with 15 ml elution buffer, collect the eluent and retain 20 μl for electrophoresis; wash the column once with 15 ml deionized water; wash the column three times with 15 ml deionized water; equilibrate the column with 20% ethanol and store.
[0056] Purified sample preparation and SDS-PAGE analysis were performed, and the results are as follows: Figure 4 As shown, SDS-PAGE analysis was performed on the supernatant, flow-through buffer, and elution buffer (i.e., purified product) during the purification process to evaluate the purification effect. M in the figure represents the standard protein molecular weight marker. Lane 1 is the supernatant protein sample, showing a complex background of protein bands, indicating the presence of various contaminating proteins in the initial sample. Lane 2 is the flow-through buffer, with a band pattern highly similar to lane 1, indicating that most non-specific contaminating proteins did not bind to the purification medium and flowed out directly. Lanes 3 and 4 (Lane 3-4) are specific elution samples, representing the first and last elution phases, respectively. The results show that after elution, a large number of contaminating protein bands visible in lanes 1 and 2 were effectively removed. A single, clear, and bright main band was visible between 60 kDa and 70 kDa, a position consistent with the theoretical molecular weight of the target protein. Comparing lanes 1 and 3, it can be seen that the purification method described in this invention can specifically enrich the target protein from the complex supernatant and obtain a high-purity elution product; the qualified purified product is selected for dialysis and concentration.
[0057] 4) Dialyze the qualified eluted sample into 1×PBS, 10% Glycerol, pH 7.4 solution, and dialyze overnight at 4°C. The target protein should be clear and no precipitate should be formed.
[0058] 5) Centrifuge at 4500 rpm and 4℃, then concentrate the target protein to a suitable concentration using an ultrafiltration tube. Samples are then analyzed by SDS-PAGE to determine the final protein concentration and quantity. The SDS-PAGE results are shown below. Figure 5 As shown, lane 1 exhibits a clear and bright main band between 60 kDa and 70 kDa, the position of which corresponds to the theoretical molecular weight of the target protein. The results indicate that after the concentration step, the target protein was effectively enriched while maintaining good integrity and purity; the final protein concentration was 2 mg / ml, the volume was 1 ml, and the protein amount was 2 mg.
[0059] The results showed that UrdA urocanate reductase was well expressed, with high purity after purification and high activity after concentration. The preparation method in this example is scalable, reproducible, and can stably obtain high-quality UrdA urocanate reductase.
[0060] This specific embodiment is merely an explanation of the present invention and is not intended to limit the present invention. Any changes made by those skilled in the art after reading the specification of the present invention, as long as they are within the scope of the claims of the present invention, will be protected by patent law.
Claims
1. A method for preparing UrdA urokanedate reductase, characterized in that, Includes the following steps: S1. The codon-optimized UrdA gene was cloned into the pSHA-2 (His-tag) expression vector to construct a recombinant expression plasmid; the sequence of the codon-optimized UrdA gene is shown in SEQ ID NO.2; S2. The recombinant expression plasmid was transformed into Rosetta(DE3) strain, and UrdA protein was expressed by IPTG induction; S3. After ultrasonic disruption of bacterial cells, the supernatant was collected and the protein was purified using Ni-Smart affinity chromatography; S4. Dialyze the purified protein; S5. The dialysate is concentrated by ultrafiltration to obtain UrdA protein.
2. The method for preparing UrdA urokanic acid reductase according to claim 1, characterized in that, In step S1, the expression vector is kanamycin resistant and its cloning site is BamHI-XhoI.
3. The method for preparing UrdA urokanedate reductase according to claim 1, characterized in that, In step S2, the method for transforming the recombinant expression plasmid is as follows: after mixing the recombinant expression plasmid with Rosetta (DE3) cells, incubate on ice, then heat shock in a 42°C water bath for 80-100 seconds, and place on ice for 4-6 minutes after heat shock.
4. The method for preparing UrdA urokanedate reductase according to claim 3, characterized in that, In step S2, the method for inducing UrdA protein expression using IPTG is as follows: Add LB liquid medium to the mixture of heat-shocked recombinant expression plasmid and Rosetta (DE3) cells, and incubate at 37°C and 200-250 rpm for 45-60 minutes; The culture was inoculated onto LB agar plates and incubated overnight at 37°C. The cultured strain was inoculated into LB liquid medium and cultured at 37°C and 220 rpm until the bacterial cell OD reached the target value. 600 The concentration was 0.4-0.
6. IPTG was added to the culture to a concentration of 0.2 mM, and the culture was continued at 16℃ and 220 rpm for 16 h to induce the expression of the fusion protein.
5. The method for preparing UrdA urokanic acid reductase according to claim 4, characterized in that, In step S3, the bacterial cells are resuspended in PBS, β-mercaptoethanol and PMSF are added, and the cells are sonicated in an ice bath for 3 seconds, paused for 3 seconds, and sonicated 90-110 times; centrifuged and the supernatant is collected.
6. The method for preparing UrdA urokanic acid reductase according to claim 5, characterized in that, In step S3, the eluent used for protein purification by Ni-Smart affinity chromatography consists of: 20 mM PB, 0.5 M NaCl, 100 mM Midazole, and pH 7.
4.
7. The method for preparing UrdA urokanic acid reductase according to claim 6, characterized in that, In step S4, the dialysis method involves adding the purified protein to a PBS solution containing glycerol at pH 7.4 and dialyzing overnight at 4°C.
8. The method for preparing UrdA urokanedate reductase according to claim 1, characterized in that, In step S5, ultrafiltration concentration is carried out at 4000-5000 rpm and 4℃.
9. UrdA urocanate reductase prepared by the preparation method according to any one of claims 1-8.
10. The application of UrdA urocanic acid reductase prepared by the preparation method according to any one of claims 1-8 in basic research, wherein the basic research includes screening of UrdA urocanic acid reductase inhibitors, research on UrdA urocanic acid reductase catalytic reactions, and research on intestinal flora metabolism-related diseases.