CN110484518B - Self-assembled short peptide tag marked fluoridase aggregate and application - Google Patents

Abstract

The invention relates to a self-assembled short peptide tag labeled fluoridase aggregate, which is prepared by combining a self-assembled short peptide tag and fluoridase. The present fluoridase aggregate is a nano-scale fluoridase utilizing self-assembled short peptide tags, which can improve catalytic efficiency, enhance thermal stability and have reusability, and can be applied to a biological conversion catalyst for fluoride, and at the same time, can be used in combination with nucleoside hydrolase to directly catalyze a substrate inorganic fluoride ion (F ^‑ ) And S-adenosyl-L-methionine (SAM), which generates 5' -FDR (fluorinated deoxyribose) and can be potentially used in the preparation of a positron emission tomography radiotracer.

Description

A self-assembled short peptide tag-labeled fluorase aggregate and its application

Technical field

The invention belongs to the technical field of protein and enzyme engineering, in particular to a self-assembled short peptide tag-labeled fluorase aggregate and its application.

Background technique

With the development of genomics and proteomics, more and more proteins are expressed and purified through genetic recombination technology and engineered bacterial strains. Therefore, the development of cheap and economical protein purification methods remains a fundamental task, and protein purification is also one of the core requirements of biotechnology. In the pharmaceutical industry, the use of traditional chromatography techniques such as ion exchange, affinity chromatography, gel filtration, and reversed-phase chromatography for purification and separation of proteins has been well developed and used. Over the years, however, the use of purification tags to purify proteins has become popular in the laboratory for many researchers. These tags are mainly based on affinity, including His tag, GST tag, maltose-binding protein (MBP) tag and chitin-binding domain (CBD) tag with intein-mediated cleavage site (IMPACT-CN), etc. These purification tags typically enable the protein of interest to reach a purity of approximately 90% or higher, which is sufficient for many experimental uses, such as determination of enzymatic properties and protein characterization. Among these purification tags, His-tag technology is indisputably the most commonly used pre-packed nickel column to bind His-tagged target proteins and ultimately achieve the purpose of purification. This method is widely used, but again, using nickel Columns also contribute to the prohibitive cost of protein purification. Therefore, in recent years, a new class of self-assembled short peptide tags have been used for column-free separation and purification of proteins and peptides. These methods can be used to quickly separate target proteins or peptides from background impurities and produce proteins or peptides with yields and purity comparable to His tag technology, but without the use of expensive nickel columns or resins, which can be significantly reduced. Cost of purified protein.

The self-assembling short peptide tag is an amphipathic short peptide with hydrophilic and hydrophobic residue sequences. After binding to the target protein, it can promote the target protein to self-assemble into nanometer-sized protein aggregates. The principle is that during the expression process of proteins with self-assembled tags, due to the intermolecular diffusion caused by the tag and the changes in some specific intermolecular forces such as van der Waals forces, hydrophobic interactions, metal coordination bonds, etc., these changes will occur. Under this, self-assembled protein aggregates with stable structures are formed. Because this self-assembled protein aggregate has relatively good properties, it has very important potential value in the fields of bioengineering and technology. This has also led to more and more researchers engaging in research in this area in the past decade.

With the deepening of research, after people discovered the characteristics of this type of self-assembled short peptide tags, they designed a type of self-assembly that can induce protein aggregation without reducing the activity of the aggregates, improving thermal stability, and being easy to purify and isolate. Short peptide tags, namely ELK16, L6KD and 18A, etc. This series of short peptide tags were first designed and discovered by Lin Zhanglin's research group at Tsinghua University. The ELK16 tag has a β-sheet structure, the 18A tag has an α-helical structure, and the L6KD tag is a surfactant similar to A structure that is not a β-sheet or α-helix. Subsequently, these three tags were first applied to industrial enzymes such as fatty acid enzyme A and xylosidase, and it was found that the activities of these enzymes were well retained or improved, and these self-assembled protein aggregates had better thermal stability. It is good, can be reused, simplifies the purification method, and reduces the cost of use, etc., so it has attracted more and more attention. At the same time, some researchers combined tag 18A with nitrilase and embedded it into calcium alginate-coated beads to prepare immobilized particles. They found that the immobilized nitrilase still retains high activity, and its activity is still high. The stability performance is about 10 times that of natural enzymes. These applications demonstrate that self-assembled short peptide tags have great potential and application value in enzyme and protein engineering.

Fluorinase (FIA) is a discovered enzyme that can catalyze inorganic fluoride ions (F ^- ) into organic molecules to form carbon-fluorine (CF) bonds to generate organic fluorides. In 2002, the research team of Professor David O'Hagan in the UK isolated the first natural fluorinase FIA (encoded by the flA gene) from Streptomyces cattleya. It can utilize inorganic fluoride ions (F ^- ) and S-adenosyl-L-methionine (SAM) to catalyze the SN ₂ bionucleophilic reaction to generate 5'-fluorinated deoxyadenosine (5'-FDA) and L- Methionine. With the study of fluorinase and its metabolic pathways, four new FIA enzymes were identified in different bacterial species from 2014 to 2016. Among them, the FIA enzyme found in Streptomyces xinghaiensis has the best enzymatic properties. These can be proved by its enzyme kinetic experimental data. The catalytic efficiency K _cat value of FIA found in Streptomycesxinghaiensis is 0.277±0.007min ^-1 , which is higher than the catalytic efficiency of the other four fluorases; and the enzyme is specific The property constant reaches 39.5±1.51mM ^-1 min ^-1 , which is much higher than other fluorinase. Therefore, using this fluorase to carry out molecular modification in order to obtain a fluorase with better properties is of very positive significance for its application.

Positron emission tomography (PET) is a medical imaging technology that has matured in recent years. It can provide three-dimensional and functional motion images and is the most advanced clinical examination imaging technology in the field of nuclear medicine. This technology scans and detects radioactive substances that have been injected or inhaled, producing images of local or whole-body organs in the human body. Therefore, PET scanning requires the preparation of radioactive tracers in advance. The radioactive tracer commonly used in clinical practice for a long time is fluoro-18 ( ¹⁸ F)-labeled fluorinated glucose (fluoro-D-glucose, FDG). However, it is prepared using chemical synthesis methods, which is expensive, pollutes the environment, and requires strict equipment and personnel familiar with the operation.

Through searching, no published patent documents related to the patent application of the present invention have been found.

Contents of the invention

The object of the present invention is to overcome the deficiencies of the prior art and provide a self-assembled short peptide tag-labeled fluorase aggregate and its application. The fluorase aggregate is a nano-level fluorase aggregate that utilizes self-assembled short peptide tags. Fluorinase can improve catalytic efficiency, enhance thermal stability and be reusable. The fluorase aggregate can be used as a bioconversion catalyst for fluoride. At the same time, the fluorase aggregate can interact with nucleosides. The combination of hydrolase directly catalyzes the substrates inorganic fluoride ion (F ^- ) and S-adenosyl-L-methionine (SAM) to generate 5'-FDR (fluorinated deoxyribose), which can be potentially used in the preparation of Radioactive tracers in positron emission tomography.

The technical solutions adopted by the present invention to solve the technical problems are:

A self-assembling short peptide tag-labeled fluorinase aggregate is prepared by combining a self-assembling short peptide tag and a fluorinase aggregate.

Moreover, the fluorinase aggregate is FIA-ELK16, the gene sequence of its encoding gene is SEQ NO.1, and the amino acid sequence of its encoding gene is SEQ NO.2;

Alternatively, the fluorinase aggregate is FIA-L6KD, the gene sequence of its encoding gene is SEQ NO.3, and the amino acid sequence of its encoding gene is SEQ NO.4;

Alternatively, the fluorinase aggregate is FIA-18A, the gene sequence of its encoding gene is SEQ NO.5, and the amino acid sequence of its encoding gene is SEQ NO.6.

Moreover, the optimal temperatures of FIA-ELK16, FIA-L6KD, and FIA-18A are 40°C, 50°C, and 60°C respectively, and the optimal pH values are all 6.0.

Moreover, the size of the FIA-ELK16 is 500-600nm; the size of the FIA-L6KD and FIA-18A are both 200-300nm.

Moreover, the substrate of the fluorase aggregate is S-adenosyl-L-methionine, that is, SAM.

Moreover, the catalytic product of the fluorinase aggregate is 5'-fluorinated deoxyadenosine, that is, 5'-FDA.

Moreover, the self-assembling short peptide tag is ELK16, L6KD or 18A.

The preparation method of self-assembled short peptide tag-labeled fluorase aggregates as described above, the steps are as follows:

⑴ Obtain the amino acid sequence of the self-assembled short peptide tag by consulting the literature, and optimize its DNA coding sequence, that is, according to the codon preference of E. coli, its DNA coding sequence is optimized and synthesized in vitro;

⑵Use overlapping PCR technology to connect the DNA sequence of the synthesized self-assembling peptide to the downstream of the DNA coding sequence of fluorinase, that is, the 3' end. The DNA sequence of fluorinase is SEQ NO.7 to form a recombinant DNA sequence;

⑶ Insert the recombinant DNA sequence into the kanamycin-resistant plasmid to construct the recombinant plasmid;

⑷Introduce the recombinant plasmid into E. coli BL21 (DE3), add LB medium containing 50 mg/mL kanamycin, and culture at 37°C until the OD ₆₀₀ value is 0.6. Then induce at 16°C and add 0.05~0.1mM IPTG to induce expression. For 24 hours, bacteria were collected;

⑸ Then break the bacteria and collect the precipitate by centrifugation. After eluting the precipitate three times with buffer solution, remove the impurities to obtain the target protein, which is the fluorase aggregate.

Application of self-assembled short peptide tag-labeled fluorase aggregates as described above in bioconversion catalysts for fluoride.

Application of self-assembled short peptide tag-labeled fluorase aggregates as described above in the preparation of radioactive tracers for positron emission tomography.

The advantages and positive effects achieved by the present invention are:

1. The fluorase aggregate of the present invention is a nano-level fluorase that utilizes self-assembled short peptide tags, which can improve catalytic efficiency, enhance thermal stability and have reusability. The fluorase aggregate can be used Neutralization of fluoride biotransformation catalysts for use in the preparation of radioactive tracers for positron emission tomography.

2. The method of the present invention applies genetic engineering methods to carry out molecular modification of soluble fluorase, and uses three self-assembled short peptide tags to construct an enzyme that can improve the catalytic efficiency of the enzyme, enhance thermal stability and make it have Reusable nanoscale fluorinase. Three plasmids were obtained through gene cloning. The three plasmids were transformed into the Escherichia coli prokaryotic expression system for efficient heterologous expression. After induced expression and purification, the fluorase aggregates were obtained.

3. Through transmission electron microscope observation, the present invention proves that the three prepared fluorase aggregates all form nanometer-level protein particles.

4. The present invention measured the enzyme kinetic parameters of three fluorase aggregates through high-performance liquid phase method, and proved that the three fluorase aggregates can catalyze S-adenosyl-L-methionine (SAM), Generates 5'-fluorodeoxyadenosine (5'-FDA). Moreover, experiments also proved that among the three fluorase aggregates, FIA-L6KD has higher catalytic efficiency, stronger thermal stability, and can be reused compared to soluble fluorase. At the same time, according to the constructed nucleoside hydrolase, a two-step enzymatic reaction was performed together with fluorase, demonstrating the potential of using isotope-labeled ¹⁸ F to synthesize 5'- ¹⁸ FDR radiotracer, which can be used for normal In electron emission tomography (PET).

Fluorine-18 ( ¹⁸ F)-labeled fluoride ions are catalyzed by fluorinase to generate 5'-18 FDA, which is then catalyzed by some enzymes (such as nucleoside hydrolase) to generate 5'- ¹⁸ FDA, which has the same effect as fluorinated ribose. Radioactive tracer 5'- ¹⁸ FDR (5'-fluorodeoxyribose, 5'-FDR). Using this biosynthesis method can not only reduce the production cost of radioactive tracers, but also be more environmentally friendly. Therefore, using three self-assembling short peptide tags to molecularly modify fluorase to obtain a fluorase with better enzymatic properties is also of great significance to the application and promotion of PET.

5. The method of the present invention prepares nanoscale fluorase aggregates by modifying the enzyme molecule level to improve the catalytic efficiency, thermal stability and reusability of the enzyme; this method uses self-assembled short peptide tags , construct nanoscale fluorase aggregates that can improve the catalytic efficiency, thermal stability and reusability of the enzyme. The three fluorase aggregates provided by the invention (FIA-ELK16, FIA-L6KD, FIA- 18A) In the presence of inorganic fluoride ions, it can catalyze S-adenosyl-L-methionine (SAM) to generate 5'-fluorodeoxyadenosine (5'-FDA).

6. The nanoscale fluorinase aggregates of the present invention can be prepared into biocatalysts for biotransformation of fluoride; the nanoscale fluorinase aggregates can also be used to prepare radioactive materials for positron emission tomography (PET). Tracer, widely used.

Description of the drawings

Figure 1 is a schematic diagram of the enzyme reactions that can be catalyzed by three self-assembling peptide-labeled fluorase aggregates FIA-ELK16, FIA-L6KD, and FIA-18A in the present invention; it can be seen that the fluorase aggregates can utilize inorganic fluoride ions ( F ^- ), catalyzes S-adenosyl-L-methionine (SAM) to generate 5'-fluorodeoxyadenosine (5'-FDA) and L-methionine;

Figure 2 is a diagram showing the results of using transmission electron microscopy to observe the particle size of three types of fluorinase aggregates FIA-ELK16, FIA-L6KD and FIA-18A in the present invention; the figure shows the particle size of three types of fluorinase aggregates at the nanometer level;

Figure 3 is a diagram showing the results of using HPLC/LC-MS to detect fluorase aggregate reaction products in the present invention; the liquid chromatogram and mass spectrum correspond to the enzyme reaction product 5'-fluorodeoxyadenosine listed in Figure 1 (5'-FDA), the arrow points to the liquid chromatography signal of the corresponding enzyme reaction product, and the structure and molecular weight of the enzyme reaction product are attached to the mass spectrum;

Figure 4 is a diagram showing the purification results of the three fluorinase aggregates FIA-ELK16, FIA-L6KD and FIA-18A in the present invention; among them, FIA-ELK16 and FIA-L6KD can be purified well, and FIA-18A can also be purified. ;

Figure 5 shows the Mieman enzyme kinetics of three fluorase aggregates FIA-ELK16, FIA-L6KD and FIA-18A in the present invention using S-adenosyl-L-methionine (SAM) as a substrate. Fitting curve graph;

Figure 6 is a diagram showing the results of verifying the reusability of three fluorase aggregates FIA-ELK16, FIA-L6KD and FIA-18A in the present invention;

Figure 7 is a diagram showing the results of the two-step enzymatic reaction between three fluorase aggregates and nucleoside hydrolase (TvNH) in the present invention; wherein, Figure 7A shows the use of high-performance liquid chromatography to detect the products of the two-step enzymatic reaction in the present invention. The result graph of adenine (AD); Figure 7B is the result graph of using liquid mass spectrometry to detect the molecular weight of adenine in the present invention; Figure 7C is the relative yield of 5'-FDR of the two-step enzymatic reaction in the present invention Result graph.

Detailed ways

The embodiments of the present invention are described in detail below. It should be noted that this embodiment is illustrative, not restrictive, and cannot be used to limit the scope of the present invention.

The raw materials used in the present invention, unless otherwise specified, are all conventional commercially available products; the methods used in the present invention, unless otherwise specified, are conventional methods in the field.

A self-assembling short peptide tag-labeled fluorinase aggregate is prepared by combining a self-assembling short peptide tag and a fluorinase aggregate.

Preferably, the fluorinase aggregate is FIA-ELK16, the gene sequence of its encoding gene is SEQ NO.1, and the amino acid sequence of its encoding gene is SEQ NO.2;

Alternatively, the fluorinase aggregate is FIA-L6KD, the gene sequence of its encoding gene is SEQ NO.3, and the amino acid sequence of its encoding gene is SEQ NO.4;

Alternatively, the fluorinase aggregate is FIA-18A, the gene sequence of its encoding gene is SEQ NO.5, and the amino acid sequence of its encoding gene is SEQ NO.6.

Preferably, the optimal temperatures of FIA-ELK16, FIA-L6KD, and FIA-18A are 40°C, 50°C, and 60°C respectively, and the optimal pH values are all 6.0.

Preferably, the size of the FIA-ELK16 is 500-600nm; the size of the FIA-L6KD and FIA-18A are both 200-300nm.

Preferably, the substrate of the fluorase aggregate is S-adenosyl-L-methionine, that is, SAM.

Preferably, the catalytic product of the fluorase aggregate is 5'-fluorodeoxyadenosine, that is, 5'-FDA.

Preferably, the self-assembling short peptide tag is ELK16, L6KD or 18A.

The preparation method of self-assembled short peptide tag-labeled fluorase aggregates as described above, the steps are as follows:

⑴ Obtain the amino acid sequence of the self-assembled short peptide tag by consulting the literature, and optimize its DNA coding sequence, that is, according to the codon preference of E. coli, its DNA coding sequence is optimized and synthesized in vitro;

⑵Use overlapping PCR technology to connect the DNA sequence of the synthesized self-assembling peptide to the downstream of the DNA coding sequence of fluorinase, that is, the 3' end. The DNA sequence of fluorinase is SEQ NO.7 to form a recombinant DNA sequence;

⑶ Insert the recombinant DNA sequence into the kanamycin-resistant plasmid to construct the recombinant plasmid;

⑷Introduce the recombinant plasmid into E. coli BL21 (DE3), add LB medium containing 50 mg/mL kanamycin, culture at 37°C until the OD value is 0.6, then induce at 16°C, add 0.05~0.1mM IPTG to induce expression 24 hours, bacteria were collected;

⑸ Then break the bacteria and collect the precipitate by centrifugation. After eluting the precipitate three times with buffer solution, remove the impurities to obtain the target protein, which is the fluorase aggregate.

The self-assembled short peptide tag-tagged fluorase aggregates as described above can be used as biotransformation catalysts for fluoride.

The self-assembled short peptide tag-labeled fluorase aggregates as described above can be applied in the preparation of radioactive tracers for positron emission tomography.

Specifically, the preparation method of self-assembled short peptide tag-labeled fluorase aggregates as described above, the specific preparation process is as follows:

(1) By reviewing the literature, we obtained the amino acid sequences of three self-assembled short peptide tags ELK16, L6KD and 18A, and optimized their DNA coding sequences, that is, based on the codon preference of E. coli, their DNA coding sequences were optimized , and synthesized in vitro.

(2) Through overlapping PCR technology, the DNA sequences of the three synthesized self-assembly peptides were connected to the downstream of the DNA coding sequence of fluorase, that is, the 3' end. The DNA sequence of fluorase is SEQ NO.7, forming Recombinant DNA sequences.

(3) Insert the recombinant DNA sequence into the kanamycin-resistant plasmid to construct a recombinant plasmid.

(4) The recombinant plasmid is introduced into E. coli BL21 (DE3), placed in LB medium containing 50 mg/mL kanamycin, cultured at 37°C until the OD ₆₀₀ value is 0.6, and then induced at 16°C, adding 0.05~0.1mM IPTG Expression was induced for 24 hours, and bacteria were collected.

(5) Then break the bacteria and collect the precipitate by centrifugation. After eluting the precipitate three times with buffer solution, the impurities are removed to obtain the target protein, which is the fluorase aggregate. The results are shown in Figure 4. The figure shows Soluble fluorase and three fluorase aggregates can be expressed and purified.

(6) Study on the size of fluorinase aggregates: Use transmission electron microscopy to observe the purified fluorinase aggregates. The results are shown in Figure 2. It can be seen that all three types of fluorinase aggregates have nanoscale sizes.

(7) Enzymatic properties study: Enzymatic studies were conducted on three fluorase aggregates and found that they can catalyze S-adenosyl-L-methionine (SAM) in the presence of KF (potassium fluoride). 5'-Fluorodeoxyadenosine (5'-FDA) and L-methionine are generated, and the results are shown in Figure 1. The high-performance liquid chromatography method was used to determine the enzymatic kinetic parameters of three fluorase aggregates. The results are shown in Figure 3, which proves that compared with soluble fluorase, the three fluorase aggregates have FIA-L6KD. The catalytic efficiency is higher.

(8) By conducting thermal stability and repeatability experiments on three types of fluorinase aggregates and soluble fluorase, the results are shown in Figure 6, which proves that FIA-L6KD among the three types of fluorinase aggregates has stronger Thermal stability; and the three fluorase aggregates can be reused and still have more than 50% catalytic activity within nine times.

Finally, the constructed nucleoside hydrolase and fluorase fluorase aggregate were used to perform a two-step enzymatic reaction, revealing the potential of using isotope-labeled ¹⁸ F to synthesize 5'- ¹⁸ FDR radioactive tracers.

The more specific operation process is as follows:

1. Determination of the sequences of three fluorase aggregates

The gene sequences of the three self-assembling peptides were connected to the downstream of the fluorase sequence through overlapping PCR technology to obtain the genes described in sequences 1, 3, and 5. The amino acid sequences of the three fluorase aggregates are as follows:

FIA-ELK16:

MSADPTQRPIIGFMSDLGTTDDSVAQCKGLMHSICPGVTVIDVCHSMTPWDVEEGARYIVDLPRFFPEGTVFATTTYPATGTETRSVAVRIKQAAKGGARGQWAGSAGGFERAEGSYIYVAPNNGLLTTVLEEHGYIEAYEVSSTKVIPERPEPTFYSREMVAIPAAHLAAGFPLSEVGRPLEDSEIVRYQPPQVEISGDTLTGVVSAIDHPYGNVWTNIHRTHLEK AGIGYGKRIKIILDDVLPFEQTLVPTFADAGEIGGVAAYLNSRGYLSLARNLASLAYPFNLKAGLKVRVETNPTPPTTPTPPTTPTPLELELKLKLELELKLK

FIA-L6KD:

MSADPTQRPIIGFMSDLGTTDDSVAQCKGLMHSICPGVTVIDVCHSMTPWDVEEGARYIVDLPRFFPEGTVFATTTYPATGTETRSVAVRIKQAAKGGARGQWAGSAGGFERAEGSYIYVAPNNGLLTTVLEEHGYIEAYEVSSTKVIPERPEPTFYSREMVAIPAAHLAAGFPLSEVGRPLEDSEIVRYQPPQVEISGDTLTGVVSAIDHPYGNVWTNIHRTHLEK AGIGYGKRIKIILDDVLPFEQTLVPTFADAGEIGGVAAYLNSRGYLSLARNLASLAYPFNLKAGLKVRVETNPTPPTTPTPPTTPTPTPLLLLLLKD

FIA-18A:

MSADPTQRPIIGFMSDLGTTDDSVAQCKGLMHSICPGVTVIDVCHSMTPWDVEEGARYIVDLPRFFPEGTVFATTTYPATGTETRSVAVRIKQAAKGGARGQWAGSAGGFERAEGSYIYVAPNNGLLTTVLEEHGYIEAYEVSSTKVIPERPEPTFYSREMVAIPAAHLAAGFPLSEVGRPLEDSEIVRYQPPQVEISGDTLTGVVSAIDHPYGNVWTNIHRTHLEK AGIGYGKRIKIILDDVLPFEQTLVPTFADAGEIGGVAAYLNSRGYLSLARNLASLAYPFNLKAGLKVRVETNPTPPTTPTPPTTPTPEWLKAFYEKVLEKLKELF.

2. Plasmid construction, overexpression and purification of three fluorase aggregates

The DNA coding sequences of the three obtained fluorase aggregates were digested by double enzymes, and the genes were inserted into plasmid vectors to construct recombinant plasmids.

The recombinant plasmid was transformed into E. coli BL21(DE3) using heat shock method. Specifically, the plasmids of the three fluorase aggregates and the transformed cells of Escherichia coli were mixed evenly and heated at 42°C for 90 seconds to transform. BL21 (DE3) cells containing three fluorase aggregate plasmids were placed into LB medium containing 50 mg/mL kanamycin until the absorbance value at 600 nm (OD ₆₀₀ ) reached about 0.6. Cool it to room temperature, add isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.05mM, and incubate at 16°C for 24 hours.

The cells were then collected, lysed, and centrifuged, and the pellet was collected and washed three times with lysis buffer to obtain three aggregates. The purified protein was then analyzed by polyacrylamide gel electrophoresis (SDS-PAGE), and then the aggregates were quantified by grayscale analysis software imageJ using fluorase as a control.

3. Detect enzyme reaction products with high performance liquid chromatography (HPLC) and liquid mass spectrometry (LC-MS)

HPLC/LC-MS was used to detect the enzymatic reaction products produced by the substrates catalyzed by three fluorase aggregates. The reaction was performed in 20mM phosphate buffer, pH 7.8. Contains fluorase aggregate (0.5mg/mL), KF (200mM) and substrate SAM (200μM), react for 30 minutes. After the reaction, trifluoroacetic acid with a mass concentration of 10% was added to the system to terminate the reaction, and then centrifuged (12000 rpm, 10 min) to remove the protein, and the supernatant was used for analysis.

4. Determination of the structure, form and size of three fluorase aggregates

Three types of fluorase aggregates were observed through transmission electron microscopy (TEM) to determine the structural state and size of the aggregates. Take 10 μL of three fluorase aggregates with a final concentration of 0.5 mg/mL and their control group soluble fluorase, and then pipet them onto the glow-discharged carbon-coated copper grid respectively, and incubate at room temperature (25 °C) for 60 seconds and blotted with filter paper. The grid was then placed on a drop of phosphotungstic acid (PTA, 2% v/v, pH 7.0) for 50 seconds. Excess PTA was removed and the samples were observed with a transmission electron microscope (TEM).

5. Enzyme kinetic experiments of three fluorinase aggregates

The enzyme reaction products were detected by high-performance liquid chromatography to determine the respective activities and enzyme kinetic constants of the three fluorase aggregates. The enzymatic reaction is activated by the addition of fluorase aggregates (final concentration 0.5 mg/mL). The reaction system is phosphate buffer (20mM, pH 7.8), in which 200mM KF is added, and the substrate concentration ranges from 0 μM to 800 μM. Subsequently, under the same substrate, the reaction products at different time points were taken and detected using a high-performance liquid chromatograph.

Based on the detection results, the enzyme reaction curves at different substrate concentrations are drawn to determine the initial speed of the enzyme reaction (based on the production rate of 5'-FDA) at different concentrations, and a Miemann enzyme kinetic curve is produced. Finally, the enzyme kinetic constants V _max , K _m , K _cat and specificity constant (specificity constant) were obtained, and the results are shown in Figure 5.

Table 1 shows the kinetic parameters of Mieman enzyme obtained by using S-adenosyl-L-methionine as the substrate for three fluorase aggregates and soluble fluorase.

6. Determination of thermal stability and reusability of three fluorase aggregates

The half-lives (t _1/2 ) of three fluorase aggregates were determined to determine their thermal stability at different temperatures. The three inclusion body fluorases were stored at different temperatures for corresponding times, and then enzymatic reactions were performed. The reaction system was phosphate buffer (20mM, pH 7.8), containing fluorase aggregates (0.5mg/mL), KF (200mM) and substrate SAM (200μM), and the reaction was carried out for 30 minutes. Subsequently, the reaction product is detected by high-performance liquid chromatography, the product concentration is determined, the enzyme activity curve is drawn, and then the half-life time of the three fluorase aggregates at different temperatures can be calculated according to the formula t _1/2 = ln2/k _d .

Since the three fluorase aggregates can be separated from the reaction products through high-speed centrifugation after the enzymatic reaction is completed, they can be reused.

Table 2 shows the half-lives (t _1/2 ) of three fluorase aggregates and soluble fluorase.

In addition, the reaction product of fluorase aggregates, 5'-FDA, can be used as a substrate to generate 5'-FDR through an enzymatic reaction under the action of nucleoside hydrolase, and other downstream substances (such as polypeptides, antibodies, affinity It has the potential to introduce isotope-labeled ¹⁸ F through bio-conjugation. This ¹⁸ F marker can be used as a radiotracer for positron emission tomography (PET).

7. Biosynthesis of 5’-FDR

The three fluorase aggregates were placed at their respective optimal temperatures and reacted for 60 minutes according to the above enzymatic reaction system. After the reaction was completed, the enzyme in the system was inactivated at 95°C for 5 minutes and centrifuged (13000r). /min, 10min), collect the reaction solution. Take 200 μL of the reaction solution, add Trypanosoma vivax nucleoside hydrolase (TvNH) with a final concentration of 0.5 mg/mL, add water to adjust the volume to 400 μL, react at 37°C for 60 minutes, and then inactivate the enzyme in the system at 95°C for 5 minutes. , collect the reaction solution by centrifugation (13000r/min, 10min) and use it for HPLC and LC/MS detection.

SAM generates 5'-FDA under the catalysis of fluorase, which then generates two reaction products, 5'-FDR and adenine (AD), under the catalysis of TvNH, and the ratio of the two products is 1:1. Since 5'-FDR does not have UV absorption, but AD has strong UV absorption at 260nm, the amount of 5'-FDR generated can be known by detecting the content of AD. The results are shown in Figure 7A. The retention time of the AD standard is about 6 minutes, and the reaction products of the four fluorases are consistent with the retention times of the standards, indicating that the four fluorases and TvNH can produce 5 after a two-step enzymatic reaction. '-FDR. In order to further verify that there is AD in the reaction product, after LC-MS detection, as shown in Figure 7B, [M+H] ⁺ =136.1, and the relative molecular mass of AD is M=135.1, and the theoretical calculation [M+H ] ⁺ =136.1, consistent with the test results. Therefore, it once again shows that TvNH can catalyze 5'-FDA to generate 5'-FDR.

At the same time, HPLC was used to quantify the reaction product AD, which is the yield of 5'-FDR. Using the soluble fluorase FIA as a control, the relative yields of 5'-FDR generated by four fluorases and TvNH through a two-step enzymatic reaction. The rate is shown in Figure 7C. It can be seen from the figure that in a certain period of time, due to the higher maximum reaction rate of FIA-18A, it produces the most 5'-FDA, resulting in a higher relative yield of 5'-FDR; the maximum reaction rate of FIA-L6KD The rate is similar to FIA, so the relative yields of 5'-FDR between the two are basically the same. This experiment also showed that SAM can be directly used to directly produce 5'-FDR through a two-step enzymatic reaction. The feasibility of using fluorinase to potentially generate [ ¹⁸ F]-fluoride for PET was verified.

Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. , the scope of the present invention is not limited to the contents disclosed in the embodiments and drawings.

sequence

The fluorinase aggregate is FIA-ELK16, and the gene sequence of its encoding gene is SEQ NO.1:

atgtctgcggacccgacccagcgcccgatcattggcttcatgtctgacctgggcactaccgacgactccgtggcgcagtgcaaaggtctgatgcactctatctgcccgggtgttaccgttatcgacgtttgccacagcatgaccccgtgggacgttgaagaaggtgctcgttacatcgttgacctgccgc gcttcttcccggagggcactgttttcgcgaccaccacctacccggcgaccggtactgaaacccgtagcgttgcggttcgcatcaaacaggcggcgaaaggcggtgcgcgtggccagtgggcgggttccgcgggtggtttcgaacgtgcggaaggttcttacatctacgttgcaccgaacaac ggcctgctgaccaccgttctggaggagcacggctacatcgaagcgtacgaagtttcttctaccaaagttatcccggaacgtccggaaccgactttctattctcgtgaaatggttgcgatcccggcagcgcacctggcagctggtttcccgctgtctgaagttggtcgtccgctggaagattctgaaatcg ttcgttatcagccgccgcaggtggaaatcagcggtgacaccctgaccggtgttgtttctgcgatcgaccatccgttcggtaacgtttggaccaacatccaccgtacccacctggaaaaagcgggtatcggttacggtaaacgtatcaaaaatcatcctggacgacgttctgccgtttgagcagaccct ggttccgaccttcgcggatgctggtgaaattggcggcgtggcagcgtatctgaactctcgtggttacctgtctctggcgcgtaacgcggcatccctggcgtatccgtttaacctgaaggcgggtctgaaagttcgtgttgaaaccaacccgacaccacctactacaccgacgccgcctaccacgccaaccccgact cctctggaattggaactgaaactgaaattagaacttgaattaaaacttaaataa

The amino acid sequence of its encoding gene is SEQ NO.2:

MSADPTQRPIIGFMSDLGTTDDSVAQCKGLMHSICPGVTVIDVCHSMTPWDVEEGARYIVDLPRFFPEGTVFATTTYPATGTETRSVAVRIKQAAKGGARGQWAGSAGGFERAEGSYIYVAPNNGLLTTVLEEHGYIEAYEVSSTKVIPERPEPTFYSREMVAIPAAHLAAGFPLSEVGRPLEDSEIVRYQPPQVEISGDTLTGVVSAIDHPYGNVWTNIHRTHLEK AGIGYGKRIKIILDDVLPFEQTLVPTFADAGEIGGVAAYLNSRGYLSLARNLASLAYPFNLKAGLKVRVETNPTPPTTPTPPTTPTPTPLELELKLKLELELKLK;

The fluorinase aggregate is FIA-L6KD, and the gene sequence of its encoding gene is SEQ NO.3:

ttcgtgttgaaaccaac ccgacccctcc aaccacacctacaccgccta cgacaccgac gccaacgccgttatactgc tgttattactgaaagattaa

The amino acid sequence of its encoding gene is SEQ NO.4:

MSADPTQRPIIGFMSDLGTTDDSVAQCKGLMHSICPGVTVIDVCHSMTPWDVEEGARYIVDLPRFFPEGTVFATTTYPATGTETRSVAVRIKQAAKGGARGQWAGSAGGFERAEGSYIYVAPNNGLLTTVLEEHGYIEAYEVSSTKVIPERPEPTFYSREMVAIPAAHLAAGFPLSEVGRPLEDSEIVRYQPPQVEISGDTLTGVVSAIDHPYGNVWTNIHRTHLEK AGIGYGKRIKIILDDVLPFEQTLVPTFADAGEIGGVAAYLNSRGYLSLARNLASLAYPFNLKAGLKVRVETNPTPPTTPTPPTTPTPTPLLLLLLKD;

The fluorinase aggregate is FIA-18A, and the gene sequence of its encoding gene is SEQ NO.5:

gttcgtgttgaaaccaac ccaacccctc cgacaacaccgacgccaccg accacg cctacacctacgccggaatggc tgaaag cattttatgaaaaagtgct g gaaaaattaaaagaactgttttaa

The amino acid sequence of its encoding gene is SEQ NO.6:

MSADPTQRPIIGFMSDLGTTDDSVAQCKGLMHSICPGVTVIDVCHSMTPWDVEEGARYIVDLPRFFPEGTVFATTTYPATGTETRSVAVRIKQAAKGGARGQWAGSAGGFERAEGSYIYVAPNNGLLTTVLEEHGYIEAYEVSSTKVIPERPEPTFYSREMVAIPAAHLAAGFPLSEVGRPLEDSEIVRYQPPQVEISGDTLTGVVSAIDHPYGNVWTNIHRTHLEK AGIGYGKRIKIILDDVLPFEQTLVPTFADAGEIGGVAAYLNSRGYLSLARNLASLAYPFNLKAGLKVRVETNPTPPTTPTPPTTPTPEWLKAFYEKVLEKLKELF.

The DNA sequence of fluorase is SEQ NO.7:

Atgtctgcggacccgacccagcgcccgatcattggcttcatgtctgacctgggcactaccgacgactccgtggcgcagtgcaaaggtctgatgcactctcttgcccgggtgttaccgttatcgacgtttgccacagcatgaccccgtgggacgttgaagaaggtgctcgttacatcgttgacctgccgc gcttcttcccggagggcactgttttcgcgaccaccacctacccggcgaccggtactgaaacccgtagcgttgcggttcgcatcaaacaggcggcgaaaggcggtgcgcgtggccagtgggcgggttccgcgggtggtttcgaacgtgcggaaggttcttacatctacgttgcaccgaacaac ggcctgctgaccaccgttctggaggagcacggctacatcgaagcgtacgaagtttcttctaccaaagttatcccggaacgtccggaaccgactttctattctcgtgaaatggttgcgatcccggcagcgcacctggcagctggtttcccgctgtctgaagttggtcgtccgctggaagattctgaaatcg ttcgttatcagccgccgcaggtggaaatcagcggtgacaccctgaccggtgttgtttctgcgatcgaccatccgttcggtaacgtttggaccaacatccaccgtacccacctggaaaaagcgggtatcggttacggtaaacgtatcaaaaatcatcctggacgacgttctgccgtttgagcagaccct ggttccgaccttcgcggatgctggtgaaattggcggcgtggcagcgtatctgaactctcgtggttacctgtctctggcgcgtaacgcggcatccctggcgtatccgtttaacctgaaggcgggtctgaaagttcgtgttgaaaccaac.

sequence list

<110> Tianjin University of Science and Technology

<120> A self-assembled short peptide tag-labeled fluorase aggregate and its application

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 999

<212>DNA/RNA

<213> The fluorase aggregate is the gene sequence of the gene encoding FIA-ELK16 (Unknown)

<400> 1

atgtctgcgg acccgaccca gcgcccgatc attggcttca tgtctgacct gggcactacc 60

gacgactccg tggcgcagtg caaaggtctg atgcactcta tctgcccggg tgttaccgtt 120

atcgacgttt gccacagcat gaccccgtgg gacgttgaag aaggtgctcg ttacatcgtt 180

gacctgccgc gcttcttccc ggagggcact gttttcgcga ccaccaccta cccggcgacc 240

ggtactgaaa cccgtagcgt tgcggttcgc atcaaacagg cggcgaaagg cggtgcgcgt 300

ggccagtggg cgggttccgc gggtggtttc gaacgtgcgg aaggttctta catctacgtt 360

gcaccgaaca acggcctgct gaccaccgtt ctggaggagc acggctacat cgaagcgtac 420

gaagtttctt ctaccaaagt tatcccggaa cgtccggaac cgactttcta ttctcgtgaa 480

atggttgcga tcccggcagc gcacctggca gctggtttcc cgctgtctga agttggtcgt 540

ccgctggaag attctgaaat cgttcgttat cagccgccgc aggtggaaat cagcggtgac 600

accctgaccg gtgttgtttc tgcgatcgac catccgttcg gtaacgtttg gaccaacatc 660

caccgtaccc acctggaaaa agcgggtatc ggttacggta aacgtatcaa aatcatcctg 720

gacgacgttc tgccgtttga gcagaccctg gttccgacct tcgcggatgc tggtgaaatt 780

ggcggcgtgg cagcgtatct gaactctcgt ggttacctgt ctctggcgcg taacgcggca 840

tccctggcgt atccgtttaa cctgaaggcg ggtctgaaag ttcgtgttga aaccaacccg 900

acaccaccta ctacaccgac gccgcctacc acgccaaccc cgactcctct ggaattggaa 960

ctgaaactga aattagaact tgaattaaaa cttaaataa 999

<210> 2

<211> 332

<212> PRT

<213> The fluorase aggregate is the amino acid sequence of the gene encoding FIA-ELK16 (Unknown)

<400> 2

Met Ser Ala Asp Pro Thr Gln Arg Pro Ile Ile Gly Phe Met Ser Asp

1 5 10 15

Leu Gly Thr Thr Asp Asp Ser Val Ala Gln Cys Lys Gly Leu Met His

20 25 30

Ser Ile Cys Pro Gly Val Thr Val Ile Asp Val Cys His Ser Met Thr

35 40 45

Pro Trp Asp Val Glu Glu Gly Ala Arg Tyr Ile Val Asp Leu Pro Arg

50 55 60

Phe Phe Pro Glu Gly Thr Val Phe Ala Thr Thr Thr Tyr Pro Ala Thr

65 70 75 80

Gly Thr Glu Thr Arg Ser Val Ala Val Arg Ile Lys Gln Ala Ala Lys

85 90 95

Gly Gly Ala Arg Gly Gln Trp Ala Gly Ser Ala Gly Gly Phe Glu Arg

100 105 110

Ala Glu Gly Ser Tyr Ile Tyr Val Ala Pro Asn Asn Gly Leu Leu Thr

115 120 125

Thr Val Leu Glu Glu His Gly Tyr Ile Glu Ala Tyr Glu Val Ser Ser

130 135 140

Thr Lys Val Ile Pro Glu Arg Pro Glu Pro Thr Phe Tyr Ser Arg Glu

145 150 155 160

Met Val Ala Ile Pro Ala Ala His Leu Ala Ala Gly Phe Pro Leu Ser

165 170 175

Glu Val Gly Arg Pro Leu Glu Asp Ser Glu Ile Val Arg Tyr Gln Pro

180 185 190

Pro Gln Val Glu Ile Ser Gly Asp Thr Leu Thr Gly Val Val Ser Ala

195 200 205

Ile Asp His Pro Tyr Gly Asn Val Trp Thr Asn Ile His Arg Thr His

210 215 220

Leu Glu Lys Ala Gly Ile Gly Tyr Gly Lys Arg Ile Lys Ile Ile Leu

225 230 235 240

Asp Asp Val Leu Pro Phe Glu Gln Thr Leu Val Pro Thr Phe Ala Asp

245 250 255

Ala Gly Glu Ile Gly Gly Val Ala Ala Tyr Leu Asn Ser Arg Gly Tyr

260 265 270

Leu Ser Leu Ala Arg Asn Leu Ala Ser Leu Ala Tyr Pro Phe Asn Leu

275 280 285

Lys Ala Gly Leu Lys Val Arg Val Glu Thr Asn Pro Thr Pro Pro Thr

290 295 300

Thr Pro Thr Pro Pro Thr Thr Pro Thr Pro Thr Pro Leu Glu Leu Glu

305 310 315 320

Leu Lys Leu Lys Leu Glu Leu Glu Leu Lys Leu Lys

325 330

<210> 3

<211> 975

<212>DNA/RNA

<213> The fluorase aggregate is the gene sequence of the gene encoding FIA-L6KD (Unknown)

<400> 3

atgtctgcgg acccgaccca gcgcccgatc attggcttca tgtctgacct gggcactacc 60

gacgactccg tggcgcagtg caaaggtctg atgcactcta tctgcccggg tgttaccgtt 120

atcgacgttt gccacagcat gaccccgtgg gacgttgaag aaggtgctcg ttacatcgtt 180

gacctgccgc gcttcttccc ggagggcact gttttcgcga ccaccaccta cccggcgacc 240

ggtactgaaa cccgtagcgt tgcggttcgc atcaaacagg cggcgaaagg cggtgcgcgt 300

ggccagtggg cgggttccgc gggtggtttc gaacgtgcgg aaggttctta catctacgtt 360

gcaccgaaca acggcctgct gaccaccgtt ctggaggagc acggctacat cgaagcgtac 420

gaagtttctt ctaccaaagt tatcccggaa cgtccggaac cgactttcta ttctcgtgaa 480

atggttgcga tcccggcagc gcacctggca gctggtttcc cgctgtctga agttggtcgt 540

ccgctggaag attctgaaat cgttcgttat cagccgccgc aggtggaaat cagcggtgac 600

accctgaccg gtgttgtttc tgcgatcgac catccgttcg gtaacgtttg gaccaacatc 660

caccgtaccc acctggaaaa agcgggtatc ggttacggta aacgtatcaa aatcatcctg 720

gacgacgttc tgccgtttga gcagaccctg gttccgacct tcgcggatgc tggtgaaatt 780

ggcggcgtgg cagcgtatct gaactctcgt ggttacctgt ctctggcgcg taacgcggca 840

tccctggcgt atccgtttaa cctgaaggcg ggtctgaaag ttcgtgttga aaccaacccg 900

acccctccaa ccacacctac accgcctacg acaccgacgc caacgccgtt actgctgtta 960

ttatactgaaag attaa 975

<210> 4

<211> 324

<212> PRT

<213> The fluorase aggregate is the amino acid sequence of the gene encoding FIA-L6KD (Unknown)

<400> 4

Met Ser Ala Asp Pro Thr Gln Arg Pro Ile Ile Gly Phe Met Ser Asp

1 5 10 15

Leu Gly Thr Thr Asp Asp Ser Val Ala Gln Cys Lys Gly Leu Met His

20 25 30

Ser Ile Cys Pro Gly Val Thr Val Ile Asp Val Cys His Ser Met Thr

35 40 45

Pro Trp Asp Val Glu Glu Gly Ala Arg Tyr Ile Val Asp Leu Pro Arg

50 55 60

Phe Phe Pro Glu Gly Thr Val Phe Ala Thr Thr Thr Tyr Pro Ala Thr

65 70 75 80

Gly Thr Glu Thr Arg Ser Val Ala Val Arg Ile Lys Gln Ala Ala Lys

85 90 95

Gly Gly Ala Arg Gly Gln Trp Ala Gly Ser Ala Gly Gly Phe Glu Arg

100 105 110

Ala Glu Gly Ser Tyr Ile Tyr Val Ala Pro Asn Asn Gly Leu Leu Thr

115 120 125

Thr Val Leu Glu Glu His Gly Tyr Ile Glu Ala Tyr Glu Val Ser Ser

130 135 140

Thr Lys Val Ile Pro Glu Arg Pro Glu Pro Thr Phe Tyr Ser Arg Glu

145 150 155 160

Met Val Ala Ile Pro Ala Ala His Leu Ala Ala Gly Phe Pro Leu Ser

165 170 175

Glu Val Gly Arg Pro Leu Glu Asp Ser Glu Ile Val Arg Tyr Gln Pro

180 185 190

Pro Gln Val Glu Ile Ser Gly Asp Thr Leu Thr Gly Val Val Ser Ala

195 200 205

Ile Asp His Pro Tyr Gly Asn Val Trp Thr Asn Ile His Arg Thr His

210 215 220

Leu Glu Lys Ala Gly Ile Gly Tyr Gly Lys Arg Ile Lys Ile Ile Leu

225 230 235 240

Asp Asp Val Leu Pro Phe Glu Gln Thr Leu Val Pro Thr Phe Ala Asp

245 250 255

Ala Gly Glu Ile Gly Gly Val Ala Ala Tyr Leu Asn Ser Arg Gly Tyr

260 265 270

Leu Ser Leu Ala Arg Asn Leu Ala Ser Leu Ala Tyr Pro Phe Asn Leu

275 280 285

Lys Ala Gly Leu Lys Val Arg Val Glu Thr Asn Pro Thr Pro Pro Thr

290 295 300

Thr Pro Thr Pro Pro Thr Thr Pro Thr Pro Thr Pro Leu Leu Leu Leu

305 310 315 320

Leu Leu Lys Asp

<210> 5

<211> 1005

<212>DNA/RNA

<213> The fluorase aggregate is the gene sequence of the gene encoding FIA-18A (Unknown)

<400> 5

atgtctgcgg acccgaccca gcgcccgatc attggcttca tgtctgacct gggcactacc 60

gacgactccg tggcgcagtg caaaggtctg atgcactcta tctgcccggg tgttaccgtt 120

atcgacgttt gccacagcat gaccccgtgg gacgttgaag aaggtgctcg ttacatcgtt 180

gacctgccgc gcttcttccc ggagggcact gttttcgcga ccaccaccta cccggcgacc 240

ggtactgaaa cccgtagcgt tgcggttcgc atcaaacagg cggcgaaagg cggtgcgcgt 300

ggccagtggg cgggttccgc gggtggtttc gaacgtgcgg aaggttctta catctacgtt 360

gcaccgaaca acggcctgct gaccaccgtt ctggaggagc acggctacat cgaagcgtac 420

gaagtttctt ctaccaaagt tatcccggaa cgtccggaac cgactttcta ttctcgtgaa 480

atggttgcga tcccggcagc gcacctggca gctggtttcc cgctgtctga agttggtcgt 540

ccgctggaag attctgaaat cgttcgttat cagccgccgc aggtggaaat cagcggtgac 600

accctgaccg gtgttgtttc tgcgatcgac catccgttcg gtaacgtttg gaccaacatc 660

caccgtaccc acctggaaaa agcgggtatc ggttacggta aacgtatcaa aatcatcctg 720

gacgacgttc tgccgtttga gcagaccctg gttccgacct tcgcggatgc tggtgaaatt 780

ggcggcgtgg cagcgtatct gaactctcgt ggttacctgt ctctggcgcg taacgcggca 840

tccctggcgt atccgtttaa cctgaaggcg ggtctgaaag ttcgtgttga aaccaaccca 900

acccctccga caacaccgac gccaccgacc acgcctacac ctacgccgga atggctgaaa 960

gcattttatg aaaaagtgct ggaaaaatta aaagaactgt tttaa 1005

<210> 6

<211> 334

<212> PRT

<213> The fluorase aggregate is the amino acid sequence of the gene encoding FIA-18A (Unknown)

<400> 6

Met Ser Ala Asp Pro Thr Gln Arg Pro Ile Ile Gly Phe Met Ser Asp

1 5 10 15

Leu Gly Thr Thr Asp Asp Ser Val Ala Gln Cys Lys Gly Leu Met His

20 25 30

Ser Ile Cys Pro Gly Val Thr Val Ile Asp Val Cys His Ser Met Thr

35 40 45

Pro Trp Asp Val Glu Glu Gly Ala Arg Tyr Ile Val Asp Leu Pro Arg

50 55 60

Phe Phe Pro Glu Gly Thr Val Phe Ala Thr Thr Thr Tyr Pro Ala Thr

65 70 75 80

Gly Thr Glu Thr Arg Ser Val Ala Val Arg Ile Lys Gln Ala Ala Lys

85 90 95

Gly Gly Ala Arg Gly Gln Trp Ala Gly Ser Ala Gly Gly Phe Glu Arg

100 105 110

Ala Glu Gly Ser Tyr Ile Tyr Val Ala Pro Asn Asn Gly Leu Leu Thr

115 120 125

Thr Val Leu Glu Glu His Gly Tyr Ile Glu Ala Tyr Glu Val Ser Ser

130 135 140

Thr Lys Val Ile Pro Glu Arg Pro Glu Pro Thr Phe Tyr Ser Arg Glu

145 150 155 160

Met Val Ala Ile Pro Ala Ala His Leu Ala Ala Gly Phe Pro Leu Ser

165 170 175

Glu Val Gly Arg Pro Leu Glu Asp Ser Glu Ile Val Arg Tyr Gln Pro

180 185 190

Pro Gln Val Glu Ile Ser Gly Asp Thr Leu Thr Gly Val Val Ser Ala

195 200 205

Ile Asp His Pro Tyr Gly Asn Val Trp Thr Asn Ile His Arg Thr His

210 215 220

Leu Glu Lys Ala Gly Ile Gly Tyr Gly Lys Arg Ile Lys Ile Ile Leu

225 230 235 240

Asp Asp Val Leu Pro Phe Glu Gln Thr Leu Val Pro Thr Phe Ala Asp

245 250 255

Ala Gly Glu Ile Gly Gly Val Ala Ala Tyr Leu Asn Ser Arg Gly Tyr

260 265 270

Leu Ser Leu Ala Arg Asn Leu Ala Ser Leu Ala Tyr Pro Phe Asn Leu

275 280 285

Lys Ala Gly Leu Lys Val Arg Val Glu Thr Asn Pro Thr Pro Pro Thr

290 295 300

Thr Pro Thr Pro Pro Thr Thr Pro Thr Pro Thr Pro Glu Trp Leu Lys

305 310 315 320

Ala Phe Tyr Glu Lys Val Leu Glu Lys Leu Lys Glu Leu Phe

325 330

<210> 7

<211> 897

<212>DNA/RNA

<213> DNA sequence of fluorase (Unknown)

<400> 7

atgtctgcgg acccgaccca gcgcccgatc attggcttca tgtctgacct gggcactacc 60

gacgactccg tggcgcagtg caaaggtctg atgcactcta tctgcccggg tgttaccgtt 120

atcgacgttt gccacagcat gaccccgtgg gacgttgaag aaggtgctcg ttacatcgtt 180

gacctgccgc gcttcttccc ggagggcact gttttcgcga ccaccaccta cccggcgacc 240

ggtactgaaa cccgtagcgt tgcggttcgc atcaaacagg cggcgaaagg cggtgcgcgt 300

ggccagtggg cgggttccgc gggtggtttc gaacgtgcgg aaggttctta catctacgtt 360

gcaccgaaca acggcctgct gaccaccgtt ctggaggagc acggctacat cgaagcgtac 420

gaagtttctt ctaccaaagt tatcccggaa cgtccggaac cgactttcta ttctcgtgaa 480

atggttgcga tcccggcagc gcacctggca gctggtttcc cgctgtctga agttggtcgt 540

ccgctggaag attctgaaat cgttcgttat cagccgccgc aggtggaaat cagcggtgac 600

accctgaccg gtgttgtttc tgcgatcgac catccgttcg gtaacgtttg gaccaacatc 660

caccgtaccc acctggaaaa agcgggtatc ggttacggta aacgtatcaa aatcatcctg 720

gacgacgttc tgccgtttga gcagaccctg gttccgacct tcgcggatgc tggtgaaatt 780

ggcggcgtgg cagcgtatct gaactctcgt ggttacctgt ctctggcgcg taacgcggca 840

tccctggcgt atccgtttaa cctgaaggcg ggtctgaaag ttcgtgttga aaccaac 897

Claims (3)

1. A self-assembled short peptide tag-labeled fluoroenzyme aggregate, characterized by: the fluoridase aggregate is prepared by combining self-assembled short peptide tags with fluoridase;

the fluoridase aggregate is FIA-ELK16, the gene sequence of the coding gene is SEQ NO.1, and the amino acid sequence of the coding gene is SEQ NO.2;

or the fluoridase aggregate is FIA-L6KD, the gene sequence of the coding gene is SEQ NO.3, and the amino acid sequence of the coding gene is SEQ NO.4;

or the fluoridase aggregate is FIA-18A, the gene sequence of the coding gene is SEQ NO.5, and the amino acid sequence of the coding gene is SEQ NO.6;

the optimal temperatures of the FIA-ELK16, the FIA-L6KD and the FIA-18A are respectively 40 ℃, 50 ℃, 60 ℃ and 6.0 respectively;

the size of the FIA-ELK16 is 500-600nm; the sizes of the FIA-L6KD and the FIA-18A are 200-300nm;

the substrate of the fluoridase aggregate is S-adenosyl-L-methionine, namely SAM;

the catalysis product of the fluoridase aggregate is 5 '-fluoridated deoxyadenosine, namely 5' -FDA;

the self-assembled short peptide tag is ELK16, L6KD or 18A;

the preparation method of the self-assembled short peptide tag-labeled fluoridase aggregate comprises the following steps:

the method comprises the steps of obtaining an amino acid sequence of a self-assembled short peptide tag by consulting a literature, optimizing a DNA coding sequence of the amino acid sequence, namely optimizing the DNA coding sequence according to the preference of escherichia coli for codons, and synthesizing the DNA coding sequence in vitro;

connecting the DNA sequence of the synthesized self-assembled peptide to the downstream, namely the 3' -end, of the DNA coding sequence of the fluoridase by an overlapping PCR technology, wherein the DNA sequence of the fluoridase is SEQ NO.7 to form a recombinant DNA sequence;

thirdly, inserting the recombinant DNA sequence into a plasmid with kanamycin resistance to construct a recombinant plasmid;

recombinant preparation of four kinds of Chinese medicinal materialsIntroducing the plasmid into Escherichia coli BL21 (DE 3), culturing in LB medium containing 50mg/mL kanamycin at 37deg.C until OD ₆₀₀ The value is 0.6, then the induction is carried out at 16 ℃, 0.05 to 0.1mM IPTG is added for induction expression for 24 hours, and bacteria are collected;

fifthly, crushing the thalli, centrifuging to collect precipitate, eluting the precipitate with a buffer solution for three times, and removing impurities to obtain target protein, namely a fluoridase aggregate;

the fluoridase aggregate can utilize inorganic fluoride F ^- Catalyzing the SAM of S-adenosyl-L-methionine to produce 5 '-fluorodeoxyadenosine 5' -FDA) and L-methionine;

the kinetic parameters of the Miman enzyme obtained by using S-adenosyl-L-methionine as a substrate of FIA-ELK16, FIA-L6KD and FIA-18A are as follows:

the half-life of FIA-ELK16, FIA-L6KD, FIA-18A (t _1/2 ) The method comprises the following steps:

the FIA-ELK16, the FIA-L6KD and the FIA-18A can be reused.

2. Use of the self-assembled short peptide tag-labeled fluoroenzyme aggregate of claim 1 in a bioconversion catalyst of fluoride.

3. Use of a self-assembled short peptide tag-labeled fluoridase aggregate according to claim 1 for the preparation of a positron emission tomography radiotracer.