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CN-122012424-A - Preparation method of UrdA urocanic acid reductase

CN122012424ACN 122012424 ACN122012424 ACN 122012424ACN-122012424-A

Abstract

The invention belongs to the field of biological medicine, and particularly discloses a preparation method of UrdA urocanic acid reductase, which comprises the steps of expression plasmid construction, expression test, amplified expression and affinity purification, protein dialysis, protein concentration and the like, and the UrdA urocanic acid reductase after concentration has the characteristics of high purity and good activity. The UrdA enzyme with definite sources, stable structure and biological activity can be constructed by the invention, so that not only can standardized experimental materials be provided for related basic research, but also a unified enzyme activity detection system can be established, the effectiveness of a regulation and control means can be evaluated, and a foundation can be laid for the development of subsequent application products. The invention has important significance for further researching UrdA urocanic acid reductase inhibitor screening and clinical transformation.

Inventors

  • YANG HONGBO
  • CAO JIATIAN
  • HUANG ZHEYONG
  • Cai shiteng
  • SONG YANAN
  • QIAN JUYING
  • GE JUNBO

Assignees

  • 复旦大学附属中山医院

Dates

Publication Date
20260512
Application Date
20260306

Claims (10)

  1. 1. A method for preparing UrdA urocanic acid reductase, which is characterized by comprising the following steps: s1, cloning UrdA genes subjected to codon optimization to a pSHA-2 (His-tag) expression vector to construct a recombinant expression plasmid, wherein the UrdA gene sequence subjected to codon optimization is shown as SEQ ID NO. 2; S2, transforming the recombinant expression plasmid into a Rosetta (DE 3) strain, and expressing UrdA proteins by IPTG induction; S3, collecting supernatant after ultrasonic breaking of thalli, and purifying protein by adopting Ni-Smart affinity chromatography; S4, dialyzing the purified protein; and S5, performing ultrafiltration concentration on the dialysate to obtain UrdA protein.
  2. 2. The method for preparing UrdA urocanic acid reductase according to claim 1, wherein in the step S1, said expression vector has kanamycin resistance, and the cloning site thereof is BamHI-XhoI.
  3. 3. The method for preparing UrdA urocanic acid reductase according to claim 1, wherein in the step S2, the recombinant expression plasmid is transformed by incubating on ice after mixing the recombinant expression plasmid with Rosetta (DE 3) cells, and then heating in a 42 ℃ water bath for 80-100S, and standing on ice for 4-6 minutes after finishing the heat shock.
  4. 4. The method for preparing UrdA urocanic acid reductase according to claim 3, wherein in the step S2, the method for inducing expression of UrdA protein by IPTG comprises the steps of: Adding LB liquid culture medium into the mixture of recombinant expression plasmid and Rosetta (DE 3) cells subjected to heat shock, and culturing at 37 ℃ for 45-60 minutes at 200-250 rpm; Cultures were inoculated onto LB solid plates and incubated overnight at 37 ℃; The cultured strain was inoculated into LB liquid medium and cultured at 37℃and 220 rpm until the cell OD 600 was 0.4-0.6, IPTG was added to the culture to a concentration of 0.2mM, and the culture was continued at 16℃and 220 rpm for 16 hours to induce the expression of the fusion protein.
  5. 5. The method for preparing UrdA urocanic acid reductase according to claim 4, wherein in step S3, the cells are resuspended in PBS, beta-mercaptoethanol and PMSF are added, the cells are broken by ice bath ultrasound, 3S is suspended by ultrasound for 3S, ultrasound is carried out for 90-110 times, and the supernatant is collected by centrifugation.
  6. 6. The method for preparing UrdA urocanic acid reductase as claimed in claim 5, wherein in the step S3, the composition of the eluent used for protein purification by Ni-Smart affinity chromatography is 20mM PB,0.5M NaCl,100 mM Imidazole,pH 7.4.
  7. 7. The method for preparing UrdA urocanic acid reductase according to claim 6, wherein in step S4, the purified protein is added to PBS solution containing glycerol at pH 7.4 and dialyzed overnight at 4 ℃.
  8. 8. The method for producing UrdA urocanic acid reductase as claimed in claim 1, wherein in step S5, ultrafiltration concentration is performed under centrifugation conditions of 4000-5000rpm and 4 ℃.
  9. 9. UrdA urocanic acid reductase produced by the method of any one of claims 1-8.
  10. 10. Use of UrdA urocanic acid reductase prepared by the method of any one of claims 1-8 in a basic study including UrdA urocanic acid reductase inhibitor screening, urdA urocanic acid reductase catalytic reaction studies, and intestinal flora metabolism-related disease studies.

Description

Preparation method of UrdA urocanic acid reductase Technical Field The invention relates to the field of biological medicine, in particular to a preparation method of UrdA urocanic acid reductase. Background With the gradual understanding of the role of intestinal flora metabolism in metabolic diseases, cardiovascular diseases and inflammation-related diseases, metabolic enzymes derived from intestinal microorganisms and the catalytic reaction processes thereof are important for research and application. Wherein, urocanic acid reductase (urocanate reductase, urdA) is used as a key enzyme in histidine metabolic pathway, can catalyze urocanic acid to generate imidazole propionic acid, and is an important node for connecting intestinal microorganism metabolic activity and host metabolic state. The existing research shows that the imidazole propionic acid has an increasing trend in various metabolic abnormal states, and the generation process of the imidazole propionic acid is highly dependent on the expression and the catalytic activity of UrdA in intestinal microorganisms. Thus, the enzymatic properties, catalytic mechanisms, activity regulation and their inhibition strategies surrounding UrdA have become key to relevant basic research, drug screening and functional intervention studies. However, current research on urocanic acid reductase has been largely dependent on natural strain expression or complex microbial culture systems. Since UrdA are mostly derived from anaerobic or facultative anaerobic microorganisms, their expression levels are significantly affected by strain background, culture conditions and environmental factors, resulting in poor reproducibility of experimental results between different studies. Meanwhile, a plurality of metabolic enzymes and interference factors often exist in a natural bacterial system, and independent, accurate and quantitative evaluation of UrdA enzyme activities is difficult. More importantly, until now, commercial reagents or standardized enzyme products of urocanic acid reductase are not available on the market, and scientific researchers cannot directly obtain purified UrdA protein for in-vitro reaction system construction, inhibitor screening, kinetic parameter determination or detection method establishment. This current situation objectively limits the in-depth development of UrdA related studies and also limits their further development in detection, intervention and transformation applications. Under the condition of lacking commercial UrdA enzyme, developing a construction method capable of repeatedly, amplifying and stably obtaining urocanic acid reductase is an urgent need in the current technical field. Disclosure of Invention The application aims to provide a method for preparing UrdA urocanic acid reductase, which solves the problems of limited enzyme sources, unstable preparation process, lack of standardized reagents and the like in the prior art, and is applied to in-vitro and in-vivo experiments such as subsequent enzyme catalytic reaction and inhibitor screening. In order to achieve the above purpose, the invention adopts the following specific technical scheme: In a first aspect, the invention provides a preparation method of UrdA urocanic acid reductase, which comprises the steps of expression plasmid construction, expression test, amplified expression and affinity purification, protein dialysis, protein concentration and the like, and the UrdA urocanic acid reductase after concentration has the characteristics of high purity and good activity. The method comprises the following specific steps: s1, cloning UrdA genes subjected to codon optimization to a pSHA-2 (His-tag) expression vector to construct a recombinant expression plasmid, wherein the UrdA gene sequence subjected to codon optimization is shown as SEQ ID NO. 2; S2, transforming the recombinant expression plasmid into a Rosetta (DE 3) strain, and expressing UrdA proteins by IPTG induction; S3, collecting supernatant after ultrasonic breaking of thalli, and purifying protein by adopting Ni-Smart affinity chromatography; S4, dialyzing the purified protein; And S5, performing ultrafiltration concentration on the dialyzate to obtain high-purity and high-activity UrdA protein. Preferably, in step S1, the expression vector has kanamycin resistance, and the cloning site thereof is BamHI-XhoI. Preferably, in the step S2, the recombinant expression plasmid is transformed by mixing the recombinant expression plasmid with Rosetta (DE 3) cells, incubating on ice, then performing heat shock in a 42 ℃ water bath for 80-100S, and standing on ice for 4-6 minutes after the heat shock is completed. Preferably, in step S2, the method for IPTG-induced expression of UrdA proteins is as follows: Adding LB liquid culture medium into the mixture of recombinant expression plasmid and Rosetta (DE 3) cells subjected to heat shock, and culturing at 37 ℃ for 45-60 minutes at 200-250 rpm; Cultures were inoculated ont