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

Self-assembled short peptide tag marked fluoridase aggregate and application Download PDF

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CN110484518B
CN110484518B CN201910601278.7A CN201910601278A CN110484518B CN 110484518 B CN110484518 B CN 110484518B CN 201910601278 A CN201910601278 A CN 201910601278A CN 110484518 B CN110484518 B CN 110484518B
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马龙
满淑丽
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Tianjin University of Science and Technology
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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

Self-assembled short peptide tag marked fluoridase aggregate and application
Technical Field
The invention belongs to the technical field of protein and enzyme engineering, and particularly relates to a self-assembled short peptide tag labeled fluoridase aggregate and application thereof.
Background
With the development of genomics and proteomics, more and more proteins are purified by gene recombination technology using the work Cheng Junchong. Therefore, development of an inexpensive and economical method for purifying proteins remains a fundamental task, and protein purification is also one of the core demands of biotechnology. In the pharmaceutical industry, purification and separation of proteins using conventional chromatographic techniques such as ion exchange, affinity chromatography, gel filtration, reverse phase chromatography, etc., have been well developed and used. However, in the laboratory, the use of purification tags to purify proteins has become popular for many researchers over the years. These tags are primarily based on affinity, including His tags, GST tags, maltose Binding Protein (MBP) tags, and Chitin Binding Domain (CBD) tags, with intein-mediated cleavage sites (IMPACT-CN), and the like. These purification tags are typically capable of achieving a protein content of interest of about 90% or more pure enough for many experimental purposes, such as measuring enzymatic properties and characterization of proteins. Among these purification tags, his-tag technology is one of the most commonly used pre-packed nickel columns to bind His-tagged target proteins, and this approach is widely used for purification purposes, but likewise, the use of nickel columns results in excessive protein purification costs. Thus, during recent years, a new class of self-assembled short peptide tags has been used for column-free separation and purification of proteins and polypeptides. These methods can be used to rapidly separate a protein or polypeptide of interest from background impurities and produce a protein or polypeptide with yields and purity comparable to His-tag technology, but without the use of expensive nickel columns or resins, etc., which can greatly reduce the cost of purifying the protein.
The self-assembled short peptide tag is an amphiphilic short peptide with a hydrophilic residue and a hydrophobic residue sequence, and can promote the self-assembly of a protein aggregate with nanometer size of a target protein after being combined with the target protein. The principle is that proteins with self-assembled tags, during expression, intermolecular diffusion due to the tags and changes in some specific intermolecular forces such as van der Waals forces, hydrophobic interactions, metal coordination bonds, etc., form self-assembled protein aggregates with stable structures under these changes. Since this self-assembled protein aggregate has relatively good properties, it has a very important potential value in the fields of biotechnology and technology, which has led more and more researchers to conduct this research in recent decades.
With the deep research, after the characteristics of the self-assembled short peptide tags are found, the self-assembled short peptide tags which can induce protein aggregation, have the advantages of no reduction of the activity of the aggregation, improved thermal stability, easiness in purification and separation and the like, namely ELK16, L6KD, 18A and the like are designed. The series of short peptide tags were designed and discovered by the university of Qinghua Lin Zhanglin subject group first, and the ELK16 tag was of a beta-sheet structure, the 18A tag was of an alpha-helix structure, and the L6KD tag was of a structure similar to a surfactant and not of a beta-sheet or alpha-helix structure. Then, these three tags were first applied to industrial enzymes such as lipase a and xylosidase, and found that the activities of these enzymes were well preserved or improved, and at the same time, these self-assembled protein aggregates have the advantages of better thermal stability, repeated use, simplified purification method, reduced use cost, and the like, and thus have been receiving more and more attention. Meanwhile, researchers combine the label 18A with nitrilase and embed the nitrilase into calcium alginate encapsulation beads to prepare immobilized particles, and the immobilized nitrilase is found to still have higher activity, and the stability of the immobilized nitrilase is about 10 times that of the natural enzyme. The application shows that the self-assembled short peptide tag has great potential and application value in enzyme and protein engineering.
Fluorinating enzyme (FIA) as an enzyme which has been found to be capable of converting inorganic fluoride ions (F - ) Catalyzing the formation of carbon-fluorine (C-F) bonds into the organic molecules to form organofluorides. In 2002, the first natural fluoride enzyme FIA (encoded by the flA gene) was isolated from Streptomyces cattleya by the group of professor David O' Hagan, uk. It can utilize inorganic fluoride ion (F) - ) And S-adenosyl-L-methionine (SAM) catalyzed SN 2 Biological nucleophilic reaction to produce 5 '-fluorodeoxyadenosine (5' -FDA) and L-methionine. With the study of the fluoridases and their metabolic pathways, four new FIA enzymes were identified in different species from 2014 to 2016. Among them, the FIA enzyme found in Streptomyces xinghiensis has optimal enzymatic properties. These can be demonstrated by its enzymatic kinetic experimental data, the catalytic efficiency K of FIA found in Streptomyces xinghiensis cat The value is 0.277+/-0.007 min -1 Catalytic efficiency of the other four fluoridasesHigher; and the specificity constant of the enzyme reaches 39.5+/-1.51 mM -1 min -1 And even more so, much higher than other fluorinated enzymes. Therefore, the molecular modification by using the fluoridase to obtain the fluoridase with better properties has positive significance for the application of the fluoridase.
Positron emission tomography (positron emission tomography, PET) is a medical imaging technology developed in recent years, which can provide three-dimensional and functional motion images, and is the most advanced clinical examination imaging technology in the nuclear medicine field. The technique generates images of local or systemic organs of the human body by scanning and detecting the injected or inhaled radiation. Therefore, the PET scanning requires the preparation of a radioactive tracer in advance, and the radioactive tracer which is commonly used clinically for a long time is fluorine-18 # 18 F) Labeled Fluoroglucose (FDG). However, it is prepared using a chemical synthesis method, is costly, is environmentally polluting, and requires strict equipment and personnel familiar with the operation.
By searching, no patent publication related to the present patent application has been found.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a self-assembled short peptide tag marked fluoridase aggregate and application thereof, wherein the fluoridase aggregate is a nano level fluoridase utilizing the self-assembled short peptide tag, can improve the catalytic efficiency, enhance the thermal stability and have reusability, can be applied to the aspect of a biological conversion catalyst of fluoride, and can be combined with nucleoside hydrolase to directly catalyze inorganic fluoride ions (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.
The technical scheme adopted for solving the technical problems is as follows:
a self-assembled short peptide tag labeled fluoridase aggregate is prepared by combining a self-assembled short peptide tag and fluoridase.
The fluoridase aggregate is FIA-ELK16, the gene sequence of the encoding gene is SEQ NO.1, and the amino acid sequence of the encoding 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.
Furthermore, the optimum temperatures of the FIA-ELK16, the FIA-L6KD and the FIA-18A are respectively 40 ℃, 50 ℃, 60 ℃ and 6.0 respectively.
Moreover, the size of the FIA-ELK16 is 500-600nm; the size of the FIA-L6KD and the size of the FIA-18A are both 200-300nm.
Furthermore, the substrate of the fluoridase aggregate is S-adenosyl-L-methionine, SAM.
Furthermore, the catalytic product of the fluoridase aggregate is 5 '-fluorodeoxyadenosine, i.e. 5' -FDA.
Moreover, 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;
introducing recombinant plasmid into Escherichia coli BL21 (DE 3), placing into LB medium containing 50mg/mL kanamycin, and culturing 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 the fluoridase aggregate.
Use of a self-assembled short peptide tag-labeled fluoroenzyme aggregate as described above in a bioconversion catalyst for fluoride.
Use of a self-assembled short peptide tag-labeled fluoroenzyme aggregate as described above for the preparation of a positron emission tomography radiotracer.
The invention has the advantages and positive effects that:
1. the fluoridase aggregate is a nanometer level fluoridase utilizing self-assembled short peptide labels, can improve catalytic efficiency, enhance thermal stability and have reusability, and can be applied to biological conversion catalysts of fluoride and radioactive tracers for preparing positron emission tomography.
2. The method of the invention adopts the method of genetic engineering (genetic engineering) to carry out molecular modification on the soluble fluorinated enzyme, and utilizes three self-assembled short peptide tags to construct the nano-scale fluorinated enzyme which can improve the catalytic efficiency of the enzyme, enhance the thermal stability and enable the enzyme to have reusability. Three plasmids are obtained by a gene cloning mode, and are respectively transformed into a prokaryotic expression system of escherichia coli for efficient heterologous expression, and then the fluoridase aggregate is obtained through induced expression and purification.
3. According to the invention, through observation of a transmission electron microscope, the prepared three fluoridase aggregates all form nano-scale protein particles.
4. The invention determines the enzyme kinetic parameters of three fluoridase aggregates by a high performance liquid phase method, and proves that the three fluoridase aggregates can catalyze S-adenosine-L-methionine (SAM) to generate 5 '-fluoridated deoxyadenosine (5' -FDA). Furthermore, experiments also prove that FIA-L6KD in the aggregate of three fluoridases, compared with soluble fluoridases,has higher catalytic efficiency, stronger thermal stability and can be recycled. Meanwhile, according to the constructed nucleoside hydrolase, two-step enzymatic reaction with a fluorinating enzyme is performed, which illustrates the use of isotope labeling 18 F Synthesis of 5 18 The potential of FDR radiotracers (radiotracers) for use in positron emission tomography (positron emission tomography, PET).
By fluorine-18% 18 F) The labeled fluoride ions are catalyzed by a fluorinating enzyme to form 5' -) 18 FDA, catalyzed by enzymes such as nucleoside hydrolases to produce a radiotracer 5' which has the same effect as fluorinated ribose 18 FDR (5 '-fluorodeoxyribose,5' -FDR). By adopting the biosynthesis method, the production cost of the radioactive tracer can be reduced, and the method is more green and environment-friendly. Therefore, three self-assembled short peptide tags are utilized to carry out molecular transformation on the fluoridase, so that the fluoridase with better enzymology property is obtained, and the method has positive significance for the application and popularization of PET.
5. According to the method, the nano-scale fluoridase aggregate is prepared by modifying the enzyme molecular level so as to improve the catalytic efficiency and the thermal stability of the enzyme and enable the enzyme to have reusability; the method utilizes self-assembled short peptide tags to construct nanoscale fluoridase aggregates which can improve the catalytic efficiency and the thermal stability of enzymes and enable the enzymes to have reusability, and the three fluoridase aggregates (FIA-ELK 16, FIA-L6KD and FIA-18A) provided by the invention can catalyze S-adenosine-L-methionine (SAM) to generate 5 '-fluorinated deoxyadenosine (5' -FDA) in the presence of inorganic fluoride ions.
6. The nano-scale fluoridase aggregate can be prepared into a biocatalyst, and is applied to bioconversion of fluoride; nanoscale fluorose aggregates can also be used to prepare radiotracers for positron emission tomography (positron emission tomography, PET), and are widely used.
Drawings
FIG. 1 is a schematic diagram of enzyme reactions that can be catalyzed by three self-assembled peptide-labeled fluoroenzyme aggregates FIA-ELK16, FIA-L6KD, FIA-18A of the present invention; it can be seen thatThe fluoridase aggregate can utilize inorganic fluoride ions (F - ) Catalyzing S-adenosyl-L-methionine (SAM) to produce 5 '-fluorodeoxyadenosine (5' -FDA) and L-methionine;
FIG. 2 is a graph showing the results of observing the particle size of three types of fluoridase aggregates FIA-ELK16, FIA-L6KD, FIA-18A using a transmission electron microscope in the present invention; the particle size of the three fluoridase aggregates is shown on the nanometer scale;
FIG. 3 is a graph showing the results of detection of the reaction product of a fluoridase aggregate using HPLC/LC-MS in the present invention; wherein the liquid chromatogram and the mass chromatogram correspond to the enzyme reaction product 5 '-fluorodeoxyadenosine (5' -FDA) listed in FIG. 1, the arrow indicates the liquid chromatogram signal of the corresponding enzyme reaction product, and the structure of the enzyme reaction product and the molecular weight thereof are attached to the mass chromatogram;
FIG. 4 is a graph showing the purification results of three fluoridase aggregates FIA-ELK16, FIA-L6KD and FIA-18A according to the invention; wherein FIA-ELK16 and FIA-L6KD can be well purified, and FIA-18A can be purified;
FIG. 5 is a graph showing a Michaelis enzyme kinetics fit obtained by using S-adenosyl-L-methionine (SAM) as a substrate for three fluoridase aggregates FIA-ELK16, FIA-L6KD and FIA-18A according to the present invention;
FIG. 6 is a graph showing the results of the present invention for verifying that three fluoridase aggregates FIA-ELK16, FIA-L6KD, FIA-18A have reusability;
FIG. 7 is a graph showing the results of a two-step enzymatic reaction of three fluoridase aggregates with nucleoside hydrolase (TvNH) in accordance with the present invention; FIG. 7A is a graph showing the results of detection of Adenine (AD) as a product of a two-step enzymatic reaction using high performance liquid chromatography in the present invention; FIG. 7B is a graph showing the results of detecting the molecular weight of adenine using a liquid chromatography-mass spectrometer in the present invention; FIG. 7C is a graph showing the relative yield of 5' -FDR from a two-step enzymatic reaction according to the invention.
Detailed Description
The following describes the embodiments of the present invention in detail, but the present embodiments are illustrative and not limitative, and are not intended to limit the scope of the present invention.
The raw materials used in the invention are conventional commercial products unless specified; the methods used in the present invention are conventional in the art unless otherwise specified.
A self-assembled short peptide tag labeled fluoridase aggregate is prepared by combining a self-assembled short peptide tag and fluoridase.
Preferably, the fluoridase aggregate is FIA-ELK16, the gene sequence of the encoding gene is SEQ NO.1, and the amino acid sequence of the encoding 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.
Preferably, the optimal temperatures of the FIA-ELK16, the FIA-L6KD and the FIA-18A are respectively 40 ℃, 50 ℃ and 60 ℃, and the optimal pH values are respectively 6.0.
Preferably, the size of the FIA-ELK16 is 500-600nm; the size of the FIA-L6KD and the size of the FIA-18A are both 200-300nm.
Preferably, the substrate of the fluoridase aggregate is S-adenosyl-L-methionine, SAM.
Preferably, the catalytic product of the fluoridase aggregate is 5 '-fluorodeoxyadenosine, i.e. 5' -FDA.
Preferably, 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;
introducing the recombinant plasmid into escherichia coli BL21 (DE 3), putting into LB culture medium containing 50mg/mL kanamycin, culturing at 37 ℃ until the OD value is 0.6, then inducing at 16 ℃, adding 0.05-0.1 mM IPTG for inducing expression for 24 hours, and collecting bacteria;
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 the fluoridase aggregate.
The self-assembled short peptide tag-labeled fluoroenzyme aggregates as described above can be used in the bioconversion catalysts of fluoride.
The self-assembled short peptide tag-labeled fluorose aggregates as described above can be used in the preparation of positron emission tomography radiotracers.
Specifically, the preparation method of the self-assembled short peptide tag-labeled fluoridase aggregate comprises the following specific preparation processes:
(1) By referring to the literature, the amino acid sequences of three self-assembled short peptide tags ELK16, L6KD and 18A are obtained, the DNA coding sequences of the three self-assembled short peptide tags ELK16, L6KD and 18A are optimized, namely, the DNA coding sequences of the three self-assembled short peptide tags ELK16, L6KD and 18A are optimized according to the preference of escherichia coli for codons, and the three self-assembled short peptide tags ELK16, L6KD and 18A are synthesized in vitro.
(2) The DNA sequences of the three synthesized self-assembled peptides are respectively connected to the downstream, namely the 3' end, of the DNA coding sequence of the fluoridase by an overlap PCR technology, and the DNA sequence of the fluoridase is SEQ NO.7 to form a recombinant DNA sequence.
(3) The recombinant DNA sequence was inserted into a kanamycin-resistant plasmid to construct a recombinant plasmid.
(4) Introducing recombinant 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 bacteria are collected after induction at 16 ℃ and induction expression is carried out for 24 hours by adding 0.05-0.1 mM IPTG.
(5) Then crushing the thalli, centrifuging to collect precipitate, eluting the precipitate with buffer solution for three times, and removing impurities to obtain target protein, namely the fluoridase aggregate, wherein the result is shown in figure 4, and the figure shows that the soluble fluoridase and the three fluoridase aggregates can be expressed and purified.
(6) Study of the size of the fluoridase aggregate: the purified fluoridase aggregate was observed by transmission electron microscopy, and as a result, as shown in FIG. 2, three kinds of fluoridase aggregates each having a nano-scale size were obtained.
(7) Enzymatic Property study: the three fluoridase aggregates were enzymatically studied and found to catalyze the production of 5 '-fluorodeoxyadenosine (5' -FDA) and L-methionine in the presence of KF (potassium fluoride) and the results are shown in FIG. 1. The results of the measurement of the enzymatic kinetic parameters of the three fluoridase aggregates by the high performance liquid phase method are shown in figure 3, which prove that the catalytic efficiency of FIA-L6KD is higher in the three fluoridase aggregates compared with that of the soluble fluoridase.
(8) The results of the thermal stability and repeatability experiments on the three types of fluoridase aggregates and the soluble fluoridase are shown in fig. 6, and the FIA-L6KD in the three types of fluoridase aggregates is proved to have stronger thermal stability; and the three fluoridase aggregates can be reused, and more than 50% of catalytic activity can be maintained within nine times.
Finally, two-step enzymatic reactions using the constructed nucleoside hydrolase and fluoridase aggregates were revealed to have the use of isotopic labeling 18 F Synthesis of 5 18 Potential of FDR radiotracers.
The more specific operation process is as follows:
1. determination of the sequence of three fluoridase aggregates
The gene sequences of the three self-assembled peptides are respectively connected to the downstream of the fluoridase sequences by an overlapping PCR technology to obtain genes as shown in the sequences 1, 3 and 5, and the amino acid sequences of the three fluoridase aggregates are as follows:
FIA-ELK16:
MSADPTQRPIIGFMSDLGTTDDSVAQCKGLMHSICPGVTVIDVCHSMTPWDVEEGARYIVDLPRFFPEGTVFATTTYPATGTETRSVAVRIKQAAKGGARGQWAGSAGGFERAEGSYIYVAPNNGLLTTVLEEHGYIEAYEVSSTKVIPERPEPTFYSREMVAIPAAHLAAGFPLSEVGRPLEDSEIVRYQPPQVEISGDTLTGVVSAIDHPYGNVWTNIHRTHLEKAGIGYGKRIKIILDDVLPFEQTLVPTFADAGEIGGVAAYLNSRGYLSLARNLASLAYPFNLKAGLKVRVETNPTPPTTPTPPTTPTPTPLELELKLKLELELKLK
FIA-L6KD:
MSADPTQRPIIGFMSDLGTTDDSVAQCKGLMHSICPGVTVIDVCHSMTPWDVEEGARYIVDLPRFFPEGTVFATTTYPATGTETRSVAVRIKQAAKGGARGQWAGSAGGFERAEGSYIYVAPNNGLLTTVLEEHGYIEAYEVSSTKVIPERPEPTFYSREMVAIPAAHLAAGFPLSEVGRPLEDSEIVRYQPPQVEISGDTLTGVVSAIDHPYGNVWTNIHRTHLEKAGIGYGKRIKIILDDVLPFEQTLVPTFADAGEIGGVAAYLNSRGYLSLARNLASLAYPFNLKAGLKVRVETNPTPPTTPTPPTTPTPTPLLLLLLKD
FIA-18A：
MSADPTQRPIIGFMSDLGTTDDSVAQCKGLMHSICPGVTVIDVCHSMTPWDVEEGARYIVDLPRFFPEGTVFATTTYPATGTETRSVAVRIKQAAKGGARGQWAGSAGGFERAEGSYIYVAPNNGLLTTVLEEHGYIEAYEVSSTKVIPERPEPTFYSREMVAIPAAHLAAGFPLSEVGRPLEDSEIVRYQPPQVEISGDTLTGVVSAIDHPYGNVWTNIHRTHLEKAGIGYGKRIKIILDDVLPFEQTLVPTFADAGEIGGVAAYLNSRGYLSLARNLASLAYPFNLKAGLKVRVETNPTPPTTPTPPTTPTPTPEWLKAFYEKVLEKLKELF。
2. plasmid construction, overexpression and purification of three fluoridase aggregates
The DNA coding sequence of the obtained three fluoridase aggregates is subjected to double enzyme digestion, and genes are inserted into a plasmid vector to construct a recombinant plasmid.
The recombinant plasmid was transformed into E.coli BL21 (DE 3) using heat shock method. Specifically, plasmid of three fluoridase aggregate and colibacillus transformed cell are mixed homogeneously and heated at 42 deg.c for 90 sec for transformation. BL21 (DE 3) cells containing three fluorinating enzyme aggregate plasmids, respectively, were placed in LB medium containing 50mg/mL kanamycin until they were cultured at 600nm (OD 600 ) The absorbance value of (2) reaches about 0.6. It was cooled to room temperature, isopropyl- β -D-thiogalactoside (IPTG) was added to a final concentration of 0.05mM, and incubated at 16℃for 24 hours.
The cells were then collected, lysed, centrifuged, the pellet collected, and washed three times with lysis buffer to obtain three aggregates. The purified proteins were then analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently quantified by grey scale analysis software imageJ against fluoridases.
3. High Performance Liquid Chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) for detecting enzyme reaction products
HPLC/LC-MS was used to detect the enzymatic reaction products of three fluoridated enzyme aggregates catalyzing substrate production. The reaction was performed in 20mM phosphate buffer pH 7.8. Comprising fluoridase aggregate (0.5 mg/mL), KF (200 mM) and substrate SAM (200. Mu.M) for 30 minutes. After the reaction was completed, trifluoroacetic acid at a mass concentration of 10% was added to the system to terminate the reaction, and the protein was removed by centrifugation (12000 rpm,10 min), and the supernatant was used for analysis.
4. Determination of the structural morphology and size of three fluoridase aggregates
Three fluoridase aggregates were observed by a Transmission Electron Microscope (TEM) to determine aggregate structure status and size. 10. Mu.L of three types of fluoridase aggregate at a final concentration of 0.5mg/mL and its control group soluble fluoridase were then separately pipetted onto glow-discharged carbon-coated copper mesh and incubated at room temperature (25 ℃) for 60 seconds and blotted dry with filter paper. The mesh 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 sample was observed with a Transmission Electron Microscope (TEM).
5. Enzyme kinetics experiments with three fluoridase aggregates
The enzyme reaction products were examined by high performance liquid phase method to determine the respective activities and enzyme kinetic constants of the three fluoridated enzyme aggregates. The enzyme reaction was activated by the addition of the fluoridated enzyme aggregate (final concentration 0.5 mg/mL). The reaction system was phosphate buffer (20 mM, pH 7.8) to which 200mM KF was added at a substrate concentration of 0. Mu.M to 800. Mu.M. Then, reaction products at different time points are taken under the same substrate and detected by using a high performance liquid chromatograph.
By examining the results, an enzyme reaction curve was drawn at different substrate concentrations to determine the initial rate of the enzyme reaction (in terms of the rate of formation by the 5' -FDA) at the different concentrations, and a Miman enzyme kinetic curve was prepared. Finally obtain V max 、K m 、K cat And a specificity constant (specificity co)nstant) these enzyme kinetic constants, the results are shown in fig. 5.
Table 1 shows the kinetic parameters of Miman' S enzyme obtained with three fluoridase aggregates and soluble fluoridase using S-adenosyl-L-methionine as substrate
6. Determination of thermal stability and reusability of three fluoridase aggregates
By measuring half-life (t) 1/2 ) To determine their thermal stability at different temperatures. At different temperatures, three inclusion body fluorinases are respectively stored for corresponding time, and then enzymatic reaction is carried out. The reaction system was phosphate buffer (20 mM, pH 7.8) containing fluoridase aggregate (0.5 mg/mL), KF (200 mM) and substrate SAM (200. Mu.M) and reacted for 30 minutes. Then, detecting the reaction product by a high performance liquid chromatograph, determining the concentration of the product, drawing an enzyme activity curve, and then according to a formula t 1/2 ＝ln2/k d Half-life times of the three fluoridase aggregates at different temperatures were calculated.
After the enzymatic reaction is finished, the three fluoridase aggregates can be separated from reaction products by high-speed centrifugation, so that the aim of repeated use is fulfilled.
Table 2 shows the half-lives (t 1/2 )。
In addition, the reaction product of the fluoridase aggregate, 5'-FDA, can be used as a substrate, and under the action of nucleoside hydrolase, the 5' -FDR is generated by enzymatic reaction, and other downstream substances (such as polypeptide, antibody, affibody molecule, etc.) are subjected to bioconjugation crosslinking (bio-conjugation) to introduce isotope labeling 18 Potential capability of F. This is 18 The F label can be used as a radio tracer (radiotracer) for positive chargingSub-emission tomography (positron emission tomography, PET).
Biosynthesis of 7.5' -FDR
The three kinds of fluoridase aggregate are respectively placed at the respective optimal temperatures, the reaction is carried out for 60min according to the enzymatic reaction system, after the reaction is finished, enzymes in the system are inactivated by 95 ℃ for 5min, and the reaction solution is collected by centrifugation (13000 r/min,10 min). 200. Mu.L of the reaction solution was taken, trypanosoma vivax nucleoside hydrolase (TvNH) was added thereto at a final concentration of 0.5mg/mL, water was added to a constant volume of 400. Mu.L, the reaction was carried out at 37℃for 60 minutes, the enzymes in the system were inactivated by 95℃for 5 minutes, and the reaction solution was collected by centrifugation (13000 r/min,10 minutes) for detection by HPLC and LC/MS.
SAM generates 5'-FDA under the catalysis of fluoridase, which then generates two reaction products 5' -FDR and Adenine (AD) under the catalysis of TvNH, with a ratio of 1:1. since 5'-FDR does not have ultraviolet absorption, but AD has strong ultraviolet absorption at 260nm, the amount of 5' -FDR produced can be determined by detecting the AD content. As shown in FIG. 7A, the retention time of the AD standard was about 6min, and the retention time of the reaction products of the four kinds of fluorinases was identical to that of the standard, which indicates that the four kinds of fluorinases and TvNH were subjected to two-step enzymatic reaction to produce 5' -FDR. To further verify the presence of AD in the reaction product, LC-MS detection was performed, as shown in FIG. 7B, where [ M+H ]] + =136.1, and AD has a relative molecular mass of m=135.1, calculated theoretically [ m+h] + =136.1, consistent with the detection result. Thus, again, tvNH is able to catalyze the production of 5'-FDR from 5' -FDA.
Meanwhile, the reaction product AD was quantified by HPLC, namely the yield of 5'-FDR, and the relative yields of the four kinds of fluorinases and TvNH to produce 5' -FDR by two-step enzymatic reaction were shown in FIG. 7C, with the soluble fluorinase FIA as a control. As can be seen from the figure, the maximum reaction rate of FIA-18A is higher over time, so that the maximum 5'-FDA is produced, resulting in a higher relative yield of 5' -FDR; the maximum reaction rate of FIA-L6KD is similar to that of FIA, so the relative yields of 5' -FDR are substantially the same. This experiment also shows that SAM can be directly utilized, through two enzymatic stepsThe reaction directly yields 5' -FDR. It was verified that potential formation by use of a fluoridase was possible [ 18 F]Fluoride, feasibility of application to PET.
Although 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 invention and the appended claims, and therefore the scope of the invention is not limited to the embodiments and the disclosure of the drawings.
Sequence(s)
The fluoridase aggregate is FIA-ELK16, and the gene sequence of the coding gene is SEQ NO.1:
atgtctgcggacccgacccagcgcccgatcattggcttcatgtctgacctgggcactaccgacgactccgtggcgcagtgcaaaggtctgatgcactctatctgcccgggtgttaccgttatcgacgtttgccacagcatgaccccgtgggacgttgaagaaggtgctcgttacatcgttgacctgccgcgcttcttcccggagggcactgttttcgcgaccaccacctacccggcgaccggtactgaaacccgtagcgttgcggttcgcatcaaacaggcggcgaaaggcggtgcgcgtggccagtgggcgggttccgcgggtggtttcgaacgtgcggaaggttcttacatctacgttgcaccgaacaacggcctgctgaccaccgttctggaggagcacggctacatcgaagcgtacgaagtttcttctaccaaagttatcccggaacgtccggaaccgactttctattctcgtgaaatggttgcgatcccggcagcgcacctggcagctggtttcccgctgtctgaagttggtcgtccgctggaagattctgaaatcgttcgttatcagccgccgcaggtggaaatcagcggtgacaccctgaccggtgttgtttctgcgatcgaccatccgttcggtaacgtttggaccaacatccaccgtacccacctggaaaaagcgggtatcggttacggtaaacgtatcaaaatcatcctggacgacgttctgccgtttgagcagaccctggttccgaccttcgcggatgctggtgaaattggcggcgtggcagcgtatctgaactctcgtggttacctgtctctggcgcgtaacgcggcatccctggcgtatccgtttaacctgaaggcgggtctgaaagttcgtgttgaaaccaacccgacaccacctactacaccgacgccgcctaccacgccaaccccgactcctctggaattggaactgaaactgaaattagaacttgaattaaaacttaaataa
the amino acid sequence of the coding gene is SEQ NO.2:
MSADPTQRPIIGFMSDLGTTDDSVAQCKGLMHSICPGVTVIDVCHSMTPWDVEEGARYIVDLPRFFPEGTVFATTTYPATGTETRSVAVRIKQAAKGGARGQWAGSAGGFERAEGSYIYVAPNNGLLTTVLEEHGYIEAYEVSSTKVIPERPEPTFYSREMVAIPAAHLAAGFPLSEVGRPLEDSEIVRYQPPQVEISGDTLTGVVSAIDHPYGNVWTNIHRTHLEKAGIGYGKRIKIILDDVLPFEQTLVPTFADAGEIGGVAAYLNSRGYLSLARNLASLAYPFNLKAGLKVRVETNPTPPTTPTPPTTPTPTPLELELKLKLELELKLK；
the fluoridase aggregate is FIA-L6KD, and the gene sequence of the coding gene is SEQ NO.3:
atgtctgcggacccgacccagcgcccgatcattggcttcatgtctgacctgggcactaccgacgactccgtggcgcagtgcaaaggtctgatgcactctatctgcccgggtgttaccgttatcgacgtttgccacagcatgaccccgtgggacgttgaagaaggtgctcgttacatcgttgacctgccgcgcttcttcccggagggcactgttttcgcgaccaccacctacccggcgaccggtactgaaacccgtagcgttgcggttcgcatcaaacaggcggcgaaaggcggtgcgcgtggccagtgggcgggttccgcgggtggtttcgaacgtgcggaaggttcttacatctacgttgcaccgaacaacggcctgctgaccaccgttctggaggagcacggctacatcgaagcgtacgaagtttcttctaccaaagttatcccggaacgtccggaaccgactttctattctcgtgaaatggttgcgatcccggcagcgcacctggcagctggtttcccgctgtctgaagttggtcgtccgctggaagattctgaaatcgttcgttatcagccgccgcaggtggaaatcagcggtgacaccctgaccggtgttgtttctgcgatcgaccatccgttcggtaacgtttggaccaacatccaccgtacccacctggaaaaagcgggtatcggttacggtaaacgtatcaaaatcatcctggacgacgttctgccgtttgagcagaccctggttccgaccttcgcggatgctggtgaaattggcggcgtggcagcgtatctgaactctcgtggttacctgtctctggcgcgtaacgcggcatccctggcgtatccgtttaacctgaaggcgggtctgaaagttcgtgttgaaaccaacccgacccctccaaccacacctacaccgcctacgacaccgacgccaacgccgttactgctgttattactgaaagattaa
the amino acid sequence of the coding gene is SEQ NO.4:
MSADPTQRPIIGFMSDLGTTDDSVAQCKGLMHSICPGVTVIDVCHSMTPWDVEEGARYIVDLPRFFPEGTVFATTTYPATGTETRSVAVRIKQAAKGGARGQWAGSAGGFERAEGSYIYVAPNNGLLTTVLEEHGYIEAYEVSSTKVIPERPEPTFYSREMVAIPAAHLAAGFPLSEVGRPLEDSEIVRYQPPQVEISGDTLTGVVSAIDHPYGNVWTNIHRTHLEKAGIGYGKRIKIILDDVLPFEQTLVPTFADAGEIGGVAAYLNSRGYLSLARNLASLAYPFNLKAGLKVRVETNPTPPTTPTPPTTPTPTPLLLLLLKD；
the fluoridase aggregate is FIA-18A, and the gene sequence of the coding gene is SEQ NO.5:
atgtctgcggacccgacccagcgcccgatcattggcttcatgtctgacctgggcactaccgacgactccgtggcgcagtgcaaaggtctgatgcactctatctgcccgggtgttaccgttatcgacgtttgccacagcatgaccccgtgggacgttgaagaaggtgctcgttacatcgttgacctgccgcgcttcttcccggagggcactgttttcgcgaccaccacctacccggcgaccggtactgaaacccgtagcgttgcggttcgcatcaaacaggcggcgaaaggcggtgcgcgtggccagtgggcgggttccgcgggtggtttcgaacgtgcggaaggttcttacatctacgttgcaccgaacaacggcctgctgaccaccgttctggaggagcacggctacatcgaagcgtacgaagtttcttctaccaaagttatcccggaacgtccggaaccgactttctattctcgtgaaatggttgcgatcccggcagcgcacctggcagctggtttcccgctgtctgaagttggtcgtccgctggaagattctgaaatcgttcgttatcagccgccgcaggtggaaatcagcggtgacaccctgaccggtgttgtttctgcgatcgaccatccgttcggtaacgtttggaccaacatccaccgtacccacctggaaaaagcgggtatcggttacggtaaacgtatcaaaatcatcctggacgacgttctgccgtttgagcagaccctggttccgaccttcgcggatgctggtgaaattggcggcgtggcagcgtatctgaactctcgtggttacctgtctctggcgcgtaacgcggcatccctggcgtatccgtttaacctgaaggcgggtctgaaagttcgtgttgaaaccaacccaacccctccgacaacaccgacgccaccgaccacgcctacacctacgccggaatggctgaaagcattttatgaaaaagtgct ggaaaaattaaaagaactgttttaa
the amino acid sequence of the coding gene is SEQ NO.6:
MSADPTQRPIIGFMSDLGTTDDSVAQCKGLMHSICPGVTVIDVCHSMTPWDVEEGARYIVDLPRFFPEGTVFATTTYPATGTETRSVAVRIKQAAKGGARGQWAGSAGGFERAEGSYIYVAPNNGLLTTVLEEHGYIEAYEVSSTKVIPERPEPTFYSREMVAIPAAHLAAGFPLSEVGRPLEDSEIVRYQPPQVEISGDTLTGVVSAIDHPYGNVWTNIHRTHLEKAGIGYGKRIKIILDDVLPFEQTLVPTFADAGEIGGVAAYLNSRGYLSLARNLASLAYPFNLKAGLKVRVETNPTPPTTPTPPTTPTPTPEWLKAFYEKVLEKLKELF。
the DNA sequence of the fluorinating enzyme is SEQ NO.7:
Atgtctgcggacccgacccagcgcccgatcattggcttcatgtctgacctgggcactaccgacgactccgtggcgcagtgcaaaggtctgatgcactctatctgcccgggtgttaccgttatcgacgtttgccacagcatgaccccgtgggacgttgaagaaggtgctcgttacatcgttgacctgccgcgcttcttcccggagggcactgttttcgcgaccaccacctacccggcgaccggtactgaaacccgtagcgttgcggttcgcatcaaacaggcggcgaaaggcggtgcgcgtggccagtgggcgggttccgcgggtggtttcgaacgtgcggaaggttcttacatctacgttgcaccgaacaacggcctgctgaccaccgttctggaggagcacggctacatcgaagcgtacgaagtttcttctaccaaagttatcccggaacgtccggaaccgactttctattctcgtgaaatggttgcgatcccggcagcgcacctggcagctggtttcccgctgtctgaagttggtcgtccgctggaagattctgaaatcgttcgttatcagccgccgcaggtggaaatcagcggtgacaccctgaccggtgttgtttctgcgatcgaccatccgttcggtaacgtttggaccaacatccaccgtacccacctggaaaaagcgggtatcggttacggtaaacgtatcaaaatcatcctggacgacgttctgccgtttgagcagaccctggttccgaccttcgcggatgctggtgaaattggcggcgtggcagcgtatctgaactctcgtggttacctgtctctggcgcgtaacgcggcatccctggcgtatccgtttaacctgaaggcgggtctgaaagttcgtgttgaaaccaac。
sequence listing
<110> university of Tianjin science and technology
<120> a self-assembled short peptide tag-labeled fluoroenzyme aggregate and use thereof
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 999
<212> DNA/RNA
<213> Gene sequence of the Gene encoding FIA-ELK16 as a fluorinating enzyme aggregate (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 fluorinating enzyme aggregate was 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> Gene sequence of the encoding Gene of FIA-L6KD as the fluorinating enzyme aggregate (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
ttactgaaag attaa 975
<210> 4
<211> 324
<212> PRT
<213> the fluorinating enzyme aggregate was 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> Gene sequence of the Gene encoding FIA-18A as the fluorinating enzyme aggregate (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 fluorinating enzyme aggregate was 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 fluorinating enzyme (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.
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