Crassostrea gigas FMO1 protein as well as preparation method and application thereof

Abstract

The invention relates to the technical field of bioengineering, in particular to crassostrea gigas FMO1 protein as well as a preparation method and application thereof. The invention provides a method for efficiently preparing crassostrea gigas FMO1 protein. According to the invention, identification of a key step of crassostrea gigas taurine synthesis, namely a key taurine metabolism gene FMO1 sequence and function generated by oxidation of sub-taurine, is realized for the first time. Therefore, the Crassostrea gigas FMO1 candidate gene sequence is accurately identified, and meanwhile, the Crassostrea gigas FMO1 in-vitro recombinant protein is efficiently and massively prepared. The invention also provides a method for detecting the activity of the crassostrea gigas FMO1 in-vitro recombinant protein for catalyzing the oxidation reaction of the hypotaurine. According to the method, the activity of the FMO1 protein and the generation of the taurine can be evaluated by monitoring the consumption of NADPH (Nicotinamide Adenine Dinucleotide Phosphate) in a reaction system.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to an FMO1 protein from the oyster *Crassostrea gigas*, its preparation method, and its applications. Background Technology

[0002] Pacific oysters are rich in taurine, making them a high-quality food source for humans to supplement natural taurine intake. Screening key genes and genetic regulatory elements involved in oyster taurine metabolism using bioinformatics and molecular biology methods, and then using these for breeding new Pacific oyster strains with high taurine content, is of great significance to the oyster farming industry. According to existing literature, taurine biosynthesis in bivalve mollusks such as oysters mainly relies on the cysteine-sulfinic acid pathway. Cysteine ​​is first converted to cysteine-sulfinic acid by cysteine ​​dioxygenase, then cysteine-sulfinic acid is converted to taurine by sulfinate alanine decarboxylase, and finally taurine is further oxidized by flavin monooxygenase (FMO) to produce taurine.

[0003] FMOs are a class of monooxygenases that use flavin adenine dinucleotide as a cofactor and depend on reduced nicotinamide adenine dinucleotide phosphate. Their core function is to catalyze the oxidation of substrates containing heteroatoms (N, S, P, Se, etc.), playing a crucial role in drug metabolism, detoxification, and the synthesis of endogenous substances. In mammals, the FMO1 gene is a key enzyme catalyzing the oxidation of taurine to taurine. The taurine oxidation reaction was proposed in the 1960s, but the enzyme catalyzing this reaction was not formally identified until 2020. Currently, there are no reports on protein preparation and enzyme activity identification of the oyster FMO1 gene. Summary of the Invention

[0004] The purpose of this invention is to provide a oyster FMO1 protein, its preparation method, and its application, in order to solve the problems existing in the prior art.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides the application of the Pacific oyster FMO1 protein in catalyzing the oxidation of taurine, wherein the Pacific oyster FMO1 protein is encoded by the nucleotide sequence shown in SEQ ID NO.1.

[0006] This invention provides the application of the Pacific oyster FMO1 protein in the preparation of products that catalyze the oxidation of taurine and / or produce taurine, wherein the Pacific oyster FMO1 protein is encoded by the nucleotide sequence shown in SEQ ID NO.1.

[0007] This invention provides a product for catalyzing the oxidation of taurine and / or producing taurine, the product comprising the oyster FMO1 protein; the oyster FMO1 protein is encoded by the nucleotide sequence shown in SEQ ID NO.1.

[0008] This invention provides a method for preparing FMO1 protein from the Pacific oyster, the method comprising the following steps: (1) Download the multiple sequence alignment hidden horse model of the FMO gene family from the pfam database, and use hmmer software to scan the predicted coding gene protein sequence set of the oyster genome to obtain the candidate FMO sequences of oyster. (2) Download the protein sequence encoded by the human FMO gene from the uniprot database, perform multiple sequence alignment between the candidate FMO sequence of the oyster and the human FMO sequence, and construct a gene phylogenetic tree using the maximum likelihood method to obtain the gene phylogenetic tree file; (3) Based on the gene phylogenetic tree file, select the candidate FMO gene of oyster that clusters with the human FMO1 gene and has the highest average expression level in the early development stage of oyster as the oyster FMO1 gene; the nucleotide sequence of the oyster FMO1 gene is shown in SEQ ID NO.1. (4) The FMO1 gene of the Pacific oyster was ligated into the pCMV-N-Myc vector by seamless cloning and then transferred into the host cell to obtain recombinant bacteria; (5) The recombinant bacteria are cultured, and the culture obtained is subjected to first centrifugation, resuspension, second centrifugation, third centrifugation and fourth centrifugation in sequence to obtain the oyster FMO1 protein.

[0009] Optionally, the genome version number of the Pacific oyster is GCA_025765685.3.

[0010] Optionally, the first centrifugation is performed at a speed of 1200 rpm for 3 minutes; The resuspension includes the step of mixing the precipitate obtained from the first centrifugation with a buffer solution to obtain a resuspension. The second centrifugation includes the step of mixing the resuspended liquid and sucrose and then performing a second centrifugation; the second centrifugation speed is 1200g and the time is 10 minutes; The third centrifugation includes the step of centrifuging the supernatant obtained from the second centrifugation; the third centrifugation speed is 100,000g, the temperature is 4℃, and the time is 60 minutes. The fourth centrifugation includes the step of mixing the microsomal precipitate obtained from the third centrifugation with the cell lysis buffer, and then performing a fourth centrifugation; the fourth centrifugation speed is 14000 rpm and the time is 5 minutes.

[0011] Optionally, the components of the buffer solution are shown in the table below: ; The cell lysis buffer includes Western and IP cell lysis buffers.

[0012] This invention provides a method for detecting the catalytic activity of taurine oxidation by FMO1 protein of Pacific oyster, comprising the steps of mixing and reacting Pacific oyster FMO1 protein obtained by the above preparation method, Tris-HCl, EDTA, taurine and NADPH, and then detecting the absorbance.

[0013] Optionally, the mixing reaction is carried out at a temperature of 37°C for 60 minutes.

[0014] Optionally, the wavelength for detecting absorbance is 340 nm.

[0015] The present invention discloses the following technical effects: This invention provides the application of the Pacific oyster FMO1 protein in catalyzing the oxidation of taurine, preparing products catalyzing the oxidation of taurine, and / or producing taurine. The Pacific oyster FMO1 protein is encoded by the nucleotide sequence shown in SEQ ID NO.1. Simultaneously, this invention provides a method for efficiently preparing the Pacific oyster FMO1 protein. A specific embodiment of this invention, using HEK293T cell line to overexpress the oyster FMO1 gene, extracts microsomes and uses a tag antibody to determine the extraction efficiency, offering the following advantages: 1) Utilizing this eukaryotic expression system ensures that oxidases such as FMO1 receive correct post-translational modifications, providing a high-quality, functionally complete enzyme source for in vitro metabolic activity studies; 2) The extraction scheme, using sucrose as a permeation protectant (maintaining microsome integrity) and centrifugation as the core, provides a standardized process for obtaining microsomes from cultured cells; 3) Concentrating the FMO1 protein using the microsome extraction method is convenient and rapid. This invention is the first to achieve the identification of the sequence and function of the key metabolic gene FMO1 in the crucial step of taurine synthesis in Pacific oysters—the oxidation of taurine to taurine. Thus, this invention accurately identifies the candidate gene sequence of Crassula ovata FMO1 and simultaneously achieves efficient and large-scale preparation of Crassula ovata FMO1 recombinant protein in vitro.

[0016] This invention also provides a method for detecting the catalytic activity of FMO1 protein in the oxidation of taurine from the Pacific oyster. It is known that FMO1 protein can catalyze the oxidation of taurine to taurine, a reaction requiring the cofactor NADPH. Therefore, the method provided by this invention can assess the activity of FMO1 and the formation of taurine by monitoring the consumption of NADPH in the reaction system. The NADPH content can be determined by the change in absorbance at A340 nm. Adding microsomes to the reaction system, compared to directly using crude cell protein extract, allows for more precise control of experimental conditions, reduces interference from irrelevant proteins, and ensures a more accurate determination of FMO1 protein activity. This not only improves the reliability of the data but also effectively reduces background noise, making the results clearer and more reliable. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 Western blot (WB) results for FMO1 protein; Figure 2 The graph shows the reaction curve of FMO1 enzyme in oysters; the vertical axis represents the amount of NADPH consumed, i.e., the amount of taurine produced; among them, the blank group (green line) indicates that no NADPH was added to the system; the H2O group (purple line) indicates that no microsomes were added to the system, and 0.1M Tris-HCl was used instead; the FMO group (yellow line) represents the experimental group. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] Example 1 1. Download the multi-sequence alignment Hidden Markov Model (HMM) file "PF00743.hmm" for the FMO gene family from the pfam database (https: / / pfam.xfam.org / ). Use the hmmsearch program in the hmmer software package to scan the predicted coding gene protein sequence set of the oyster genome GCA_025765685.3 in the NCBI database. The command is "hmmsearch --cpu 4 --notextw -E 1e-20 --domE 1e-20 --incE 1e-30 --incdomE 1e-30 --max -o Out1.txtPF00743.hmm C.gigas.protein.fasta" to obtain the oyster FMO candidate sequence.

[0025] 2. Download the protein sequence encoded by the human FMO gene from the uniprot database (https: / / www.uniprot.org / ). Merge the candidate oyster FMO sequence obtained in step 1 with the human FMO sequence into a single fasta file named "CgiCanFMO.fa". Perform multiple sequence alignment using the clustalo program, with the command: "clustalo --threads=4 --in CgiCanFMO.fa --outfmt=fa --output-order=input-order --out CgiCanFMO.aln.fa". Construct a phylogenetic tree using the fasttree program with the maximum likelihood method, with the command: "fasttree".<CgiCanFMO.aln.fa> CgiCanFMO.aln.nwk”.

[0026] 3. Based on the phylogenetic tree file "CgiCanFMO.aln.nwk" generated in step 3, three candidate genes clustered with the human FMO1 gene were identified. Subsequently, the mRNA expression profile sequencing file set (PRJNA146329) for early developmental stages of the Pacific oyster was downloaded from NCBI. Expression levels at 36 early developmental stages were calculated using the RSEM program, and then the arithmetic mean of these values ​​was calculated. The candidate FMO gene with the highest mean was selected as the Pacific oyster FMO1 gene. The obtained Pacific oyster FMO1 gene is designated "CGIg08399," and its nucleotide sequence is shown in SEQ ID NO.1, specifically:

[0027] 4. Plasmid construction: 1) Using cDNA obtained by reverse transcription of RNA extracted from oyster gill tissue as a template, the oyster FMO1 gene was amplified using primers with homologous arms of the pCMV-N-Myc plasmid. The primer sequences were: forward primer F: 5'-tccaagcttctgcaggaattcATGACGTCAGAGAAGCGGGTG-3' (SEQ ID NO.2); reverse primer R: 5'-tctgtcgacgatatcgaattcTCACCTCATGAAAAGTAAATGTACTGTAA-3' (SEQ ID NO.3).

[0028] 2) The oyster FMO1 gene was ligated into the pCMV-Myc-N vector using the seamless cloning ligation method (i.e., using the Novizan ClonExpress II One StepCloning kit and following the instructions).

[0029] 5. HEK293T cell overexpression: 1) Plasmid transformation and amplification: The ligated plasmid was transformed into E. coli DH5α competent cells, positive bacterial strains were selected and plasmids were extracted by alkaline lysis, and the plasmid concentration and quality were further determined by a nucleic acid detector.

[0030] 2) Plasmid transfection: The plasmid was transfected into HEK293T cells cultured in 10cm culture dishes at approximately 80% confluency. Transfection system: Mixture 1: 13750 ng plasmid, 27.5 μL P3000, 687.5 μL Opti-MEM; Mixture 2: 20.63 μL Lipofectamine 3000 Reagent, 687.5 μL Opti-MEM. Mixtures 1 and 2 were combined and incubated at room temperature for 10 minutes, then gently added to 10cm culture dishes and incubated at 37°C with 5% CO2.

[0031] 6. Microparticle extraction: 1) Cell collection: Microsomes were extracted 36 hours after plasmid transfection. After washing adherent cells with pre-chilled PBS, 5 mL of pre-chilled PBS was added, and cells were gently scraped off with a cell scraper and centrifuged at 1200 rpm for 3 minutes.

[0032] 2) To approximately 10 6 Add 2 mL of buffer A to each cell. The components of buffer A are shown in Table 1.

[0033] Table 1 Components of Buffer A Grind in an ice bath for 15 minutes using a pre-cooled glass homogenizer, grinding up and down 10 times every 5 minutes.

[0034] 3) Add 1M sucrose solution to the mixture to make the final sucrose concentration 250mM, centrifuge at 1200g for 10 minutes, and collect the supernatant.

[0035] 4) Centrifuge 100,000g of the supernatant at 4℃ for 60 minutes. The resulting precipitate is the microparticle precipitate.

[0036] 5) Mix the obtained microsomal precipitate with 200 μL of Western and IP cell lysis buffer, centrifuge at 14000 rpm for 5 minutes, and collect the supernatant, which is the microsomal protein solution.

[0037] 7. Cell protein extraction 1) Cell collection: 36 hours after plasmid transfection, wash adherent cells with pre-cooled PBS, add 5 mL of pre-cooled PBS, gently scrape off the cells with a cell scraper, and centrifuge at 1200 rpm for 3 minutes.

[0038] 2) Add 1 mL of Western and IP cell lysis buffer to the cell pellet, centrifuge at 14000 rpm for 5 minutes, and collect the supernatant, which is the crude protein extract.

[0039] 8. Protein concentration normalization The protein concentrations of crude cell protein extract and microsomal protein solution were detected using Nanodrop and normalized.

[0040] 9. Detection of FMO1 recombinant protein: 1) Preparation of Western Blotting Samples. Take 30 μL of crude cell protein extract and 30 μL of microsomal protein solution, add 10 μL of 4× loading buffer to each, and heat at 100℃ for 5 minutes to denature the proteins.

[0041] 2) After SDS-PAGE electrophoresis, transfer the membrane, block for 1 hour, incubate overnight at 4°C with myc tag antibody, and the next day incubate with secondary antibody at 4°C for 4 hours before development.

[0042] 3) Find specific bands corresponding to the expected molecular weight (61.6 kDa) of the target FMO1 protein. The microbody yield can be assessed based on the brightness and size of the bands.

[0043] The results are as follows Figure 1As shown in the figure. The results showed that after incubation with anti-myc antibody, specific bands corresponding to the expected molecular weight (61.6 kDa) of the target FMO1 protein were detected in both the crude cell protein extract (Cell) and the microsomal protein extract (Microsomes). Based on the band size and brightness, it can be seen that the FMO1 protein in the microsomal protein extract was significantly more abundant than that in the crude cell protein extract.

[0044] 10. FMO1 taurine oxidase activity assay: 1) Prepare the mixture according to the system in Table 2. In the system, FMO1 uses taurine as a substrate and NADPH as a cofactor to catalyze the production of taurine.

[0045] Table 2 Specific components of the mixture Note: EDTA needs to be aerated by shaking at 37℃ for 10 minutes before use. An experimental control group (without NADPH) and a control group (without microparticles) were set up.

[0046] 2) After the reaction system is mixed, it is reacted in an ELISA reader at 37℃ for 60 minutes, and the change in absorbance at 340nm is detected.

[0047] The results are as follows Figure 2As shown in the figure. The results show that the absorbance of the blank group (green curve) hardly changed, remaining stable at around 0.2. The blank group was set up without the addition of NADPH, indicating that in the absence of cofactors, no NADPH-dependent oxidation reaction occurs in the system, and therefore no NADPH is consumed, hence the almost unchanged absorbance at 340 nm. The absorbance of the control group (purple curve) remained stable at around 1.3, showing almost no decrease. The control group did not add microsomes (i.e., it did not contain FMO1 enzyme), but contained NADPH and substrate. This indicates that in the absence of FMO1 enzyme, NADPH is not consumed, further proving that the change in absorbance depends on the catalytic reaction of FMO1 enzyme, rather than the spontaneous reaction of substrate or other components. The absorbance of the experimental group (yellow curve) decreased continuously from about 1.4 to 1.0, showing a significant time-dependent decrease. The experimental group contained a complete enzyme, substrate, and cofactor system. FMO1 catalyzes the reaction with taurine as the substrate and NADPH as the cofactor, a process that consumes NADPH. Because NADPH exhibits characteristic absorption at 340 nm, its depletion directly leads to a decrease in absorbance. This result demonstrates that the FMO1 protein possesses catalytic activity for the oxidation of taurine in this system. Therefore, the FMO1 protein can catalyze the production of taurine using taurine as a substrate and NADPH as a cofactor, and the enzyme activity can be quantitatively reflected by the decrease in absorbance at 340 nm. The results from the blank and control groups also validated the enzyme-dependent and NADPH-dependent nature of this reaction, eliminating interference from non-specific reactions.

[0048] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of the FMO1 protein from the Pacific oyster in catalyzing the oxidation of taurine, characterized in that, The FMO1 protein of the oyster is encoded by the nucleotide sequence shown in SEQ ID NO.

1.

2. The application of the FMO1 protein from the Pacific oyster in the preparation of products catalyzing the oxidation of taurine and / or producing taurine, characterized in that, The FMO1 protein of the oyster is encoded by the nucleotide sequence shown in SEQ ID NO.

1.

3. A product for catalyzing the oxidation of taurine and / or producing taurine, characterized in that, The product includes the oyster FMO1 protein; the oyster FMO1 protein is encoded by the nucleotide sequence shown in SEQ ID NO.

1.

4. A method for preparing FMO1 protein from the Pacific oyster, characterized in that, The preparation method includes the following steps: (1) Download the multiple sequence alignment hidden horse model of the FMO gene family from the pfam database, and use hmmer software to scan the predicted coding gene protein sequence set of the oyster genome to obtain the candidate FMO sequences of oyster. (2) Download the protein sequence encoded by the human FMO gene from the uniprot database, perform multiple sequence alignment between the candidate FMO sequence of the oyster and the human FMO sequence, and construct a gene phylogenetic tree using the maximum likelihood method to obtain the gene phylogenetic tree file; (3) Based on the gene phylogenetic tree file, select the candidate FMO gene of oyster that clusters with the human FMO1 gene and has the highest average expression level in the early development stage of oyster as the oyster FMO1 gene; the nucleotide sequence of the oyster FMO1 gene is shown in SEQ ID NO.

1. (4) The FMO1 gene of the Pacific oyster was ligated into the pCMV-N-Myc vector by seamless cloning and then transferred into the host cell to obtain recombinant bacteria; (5) The recombinant bacteria are cultured, and the culture obtained is subjected to first centrifugation, resuspension, second centrifugation, third centrifugation and fourth centrifugation in sequence to obtain the oyster FMO1 protein.

5. The preparation method according to claim 4, characterized in that, The genome version number of the Pacific oyster is GCA_025765685.

3.

6. The preparation method according to claim 4, characterized in that, The first centrifugation was performed at a speed of 1200 rpm for 3 minutes. The resuspension includes the step of mixing the precipitate obtained from the first centrifugation with a buffer solution to obtain a resuspension. The second centrifugation includes the step of mixing the resuspended liquid and sucrose and then performing a second centrifugation; the second centrifugation speed is 1200g and the time is 10 minutes; The third centrifugation includes the step of centrifuging the supernatant obtained from the second centrifugation; the third centrifugation speed is 100,000g, the temperature is 4℃, and the time is 60 minutes. The fourth centrifugation includes the step of mixing the microsomal precipitate obtained from the third centrifugation with the cell lysis buffer, and then performing a fourth centrifugation; the fourth centrifugation speed is 14000 rpm and the time is 5 minutes.

7. The preparation method according to claim 6, characterized in that, The components of the buffer solution are shown in the table below: ； The cell lysis buffer includes Western and IP cell lysis buffers.

8. A method for detecting the catalytic activity of FMO1 protein in the Pacific oyster for the oxidation of taurine, characterized in that, The method includes the step of reacting the oyster FMO1 protein obtained by the preparation method according to any one of claims 4-7 with Tris-HCl, EDTA, taurine and NADPH, and then detecting the absorbance.

9. The method according to claim 8, characterized in that, The mixing reaction was carried out at a temperature of 37°C for 60 minutes.

10. The method according to claim 8, characterized in that, The wavelength for detecting absorbance is 340 nm.

Images