CN110484518A - A kind of fluorination enzyme aggregate of self-assembled short peptide label label and application - Google Patents
A kind of fluorination enzyme aggregate of self-assembled short peptide label label and application Download PDFInfo
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- CN110484518A CN110484518A CN201910601278.7A CN201910601278A CN110484518A CN 110484518 A CN110484518 A CN 110484518A CN 201910601278 A CN201910601278 A CN 201910601278A CN 110484518 A CN110484518 A CN 110484518A
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- fluorination
- enzyme
- label
- self
- short peptide
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Abstract
The present invention relates to a kind of fluorination enzyme aggregate of self-assembled short peptide label label, the fluorination enzyme aggregate is combined and is prepared by self-assembled short peptide label and fluorination enzyme.This fluorination enzyme aggregate is a kind of fluorination enzyme of Nano grade using self-assembled short peptide label, it can be improved catalytic efficiency, enhance thermal stability and there is reusability, the fluorination enzyme aggregate can be applied in terms of the biotransformation catalyst of fluoride, simultaneously, the fluorination enzyme aggregate can be combined with nucleoside hydrolase, direct catalysis substrate inorganic fluorion (F‑) and s-adenosyl-L-methionine (SAM), generate 5 '-FDR (fluorination deoxyribose), so can potential application in the radioactive tracer for preparing positron emission computerized tomography.
Description
Technical field
The invention belongs to protein and technical field of enzyme engineering, especially a kind of fluorination enzyme of self-assembled short peptide label label
Aggregation and application.
Background technique
With the development of genomics and proteomics, more and more protein are used by gene recombination technology
Engineering strain carries out expression and purification.So the exploitation of cheap, economic method of purifying protein is still a basic task,
Protein purification is similarly one of the core requirement of biotechnology.In pharmaceutical industry, using traditional chromatographic technique such as from
The purifies and separates protein such as sub- exchange, affinity chromatography, gel filtration and reverse-phase chromatography, are developed and are used well.So
And in these years, in the lab, for many researchers, ten shuntings are become come protein purification using purification tag
Row.These labels are based primarily upon affinity, including His label, GST label, maltose-binding protein (MBP) label and chitin
Binding domain (CBD) label and include peptide-mediated cleavage site (IMPACT-CN) etc..These purification tags usually can make purpose
Protein content reaches about 90% or higher purity, and such purity is sufficient to many experimental uses, such as measurement zymetology
The characterization of property and protein.In these purification tags, His-tag technology is a kind of indisputable most common pre-filled nickel
Column carrys out the target protein of junction belt His label, is finally reached the purpose of purifying, and this method is widely used, but same, uses
It is excessively high that nickel column also results in protein purification expense.Therefore, between in recent years, a new class of self-assembled short peptide label is used for albumen
Matter and polypeptide without column separating purification.These methods can be used for rapidly separating target protein or polypeptide with background impurities,
And generate there is protein or polypeptide with the comparable yield of His label technique and purity, but not using expensive nickel column or
The cost of purifying protein can be greatly lowered in resin etc..
Self-assembled short peptide label is a kind of amphiphilic small peptide with hydrophilic residue and hydrophobic residue sequence, with mesh
Protein binding after, can promote destination protein self assembly have nano-scale protein aggregate.Wherein principle is that have
The protein of self assembly label, during expression, the intermolecular diffusion as caused by label and some specific intermolecular
Change of active force such as Van der Waals force, hydrophobic effect, metal coordinate bond etc., so that foring has surely under these changes
Determine the self-assembled protein aggregation of structure.Since this self-assembled protein aggregation has better property, so
It has highly important potential value in bioengineering and technical field, this also allows between last decade, more and more to study
Personnel are engaged in the research of this respect.
With going deep into for research, it has been found that after the characteristics of this kind of self-assembled short peptide label, devise one kind can
The activity of inducible protein aggregation and aggregation does not reduce, thermal stability improves, is easy to the self assembly of many advantages, such as purifies and separates
Small peptide label, i.e. ELK16, L6KD and 18A etc..A series of this small peptide label at first by Lin Zhanglin seminar, Tsinghua University design and
It was found that and ELK16 label is β-lamellar structure, 18A label is α-helixstructure, and L6KD label is a kind of and surfactant
It is similar, and it is not belonging to the structure of β-lamella or alpha-helix.Then, these three labels are taken the lead in being applied to fatty acid enzyme A, xylose
The industrial enzymes such as glycosides enzyme, and find that the activity of these enzymes is retained or increased well, while these self assembly albumen
Aggregation has many advantages, such as that thermal stability is more preferable, can reuse and simplify purification process, reduces use cost, from
And it receives people and more and more pays close attention to.Meanwhile there is researcher to be embedded into label 18A in conjunction with nitrilase, and by it
In calcium alginate encapsulated pearl, it is prepared into immobilization particle, the nitrilase after finding immobilization still retains higher activity, stablizes
Performance reaches 10 times of native enzyme or so.These applications are all shown, and self-assembled short peptide label has in enzyme and protein engineering
There are huge potentiality and application value.
Fluorination enzyme (FIA), can be by inorganic fluorion (F as a kind of enzyme having found-) be catalyzed into organic molecule
Carbon-fluorine (C-F) key is formed, organic fluoride is generated.In 2002, the research group of Britain David O ' Hagan professor from
First natural fluorination enzyme FIA (being encoded by flA gene) is isolated in Streptomyces cattleya.It can utilize nothing
Machine fluorine ion (F-) and s-adenosyl-L-methionine (SAM) catalysis SN2Biological necleophilic reaction generates 5 '-fluorination desoxyadenossines
(5 '-FDA) and L-Methionine.With the research to fluorination enzyme and its metabolic pathway, from 2014 to 2016 year, in different strain
In four new FIA enzymes have been determined.Wherein, the FIA enzyme found in Streptomycesxinghaiensis has optimal
Zymologic property.These can be proved by its enzyme kinetics experimental data, be sent out in Streptomycesxinghaiensis
Existing FIA, catalytic efficiency KcatValue is 0.277 ± 0.007min-1, than other four kinds be fluorinated enzymes catalytic efficiency it is higher；And
The specificity constant of enzyme reaches 39.5 ± 1.51mM-1min-1, even more much higher than other fluorination enzymes.Therefore, using this fluorination enzyme into
Row molecular modification has very positive meaning for its application in the hope of obtaining the more excellent fluorination enzyme of property.
Positron emission computerized tomography (positron emission tomography, PET) is mature in recent years
A medical imaging technology, it is capable of providing the image of three peacekeeping functional movements, is the state-of-the-art clinical inspection of the field of nuclear medicine
Look into image technology.The radiation that the technology is injected into or is inhaled by Scanning Detction generates body local or systemic organs
Image.Therefore, PET scan needs previously prepared radioactive tracer, and it is fluoro- that clinic, which does common radiotracer, for a long time
18(18F) the fluorination glucose (fluoro-D-glucose, FDG) marked.But it is prepared using chemical synthesis process, cost
It is higher, there is pollution to environment, and need stringent equipment and be familiar with the personnel operated.
By retrieval, patent publication us relevant to present patent application is not yet found.
Summary of the invention
Place that the purpose of the present invention is to overcome the deficiency in the prior art provides a kind of fluorination of self-assembled short peptide label label
Enzyme aggregate and application, the fluorination enzyme aggregate are a kind of fluorination enzyme of Nano grade using self-assembled short peptide label, energy
Catalytic efficiency is enough improved, thermal stability is enhanced and there is reusability, which can apply in fluoride
Biotransformation catalyst in terms of in, meanwhile, which can be combined with nucleoside hydrolase, direct catalysis substrate without
Machine fluorine ion (F-) and s-adenosyl-L-methionine (SAM), 5 '-FDR (fluorination deoxyribose) is generated, and then potential can answer
In the radioactive tracer for preparing positron emission computerized tomography.
The technical solution adopted by the present invention to solve the technical problems is:
A kind of fluorination enzyme aggregate of self-assembled short peptide label label, the fluorination enzyme aggregate is to pass through self-assembled short peptide
Label and fluorination enzyme are combined and are prepared.
Moreover, the fluorination enzyme aggregate is FIA-ELK16, the gene order of encoding gene is SEQ NO.1, is compiled
The amino acid sequence of code gene is SEQ NO.2；
Alternatively, the fluorination enzyme aggregate is FIA-L6KD, the gene order of encoding gene is SEQ NO.3, coding
The amino acid sequence of gene is SEQ NO.4；
Alternatively, the fluorination enzyme aggregate is FIA-18A, the gene order of encoding gene is SEQ NO.5, coding
The amino acid sequence of gene is SEQ NO.6.
Moreover, the optimum temperature of described FIA-ELK16, FIA-L6KD, FIA-18A are respectively 40 DEG C, 50 DEG C, 60 DEG C, most
Suitable pH value is 6.0.
Moreover, the size of the FIA-ELK16 is 500-600nm；The size of the FIA-L6KD and FIA-18A is
200-300nm。
Moreover, the substrate of the fluorination enzyme aggregate is s-adenosyl-L-methionine, i.e. SAM.
Moreover, the catalysate of the fluorination enzyme aggregate is 5 '-fluorination desoxyadenossines, i.e. 5 '-FDA.
Moreover, the self-assembled short peptide label is ELK16, L6KD or 18A.
The preparation method of the fluorination enzyme aggregate of self-assembled short peptide label label as described above, steps are as follows:
(1) by consulting literatures, the amino acid sequence of self-assembled short peptide label is obtained, by its DNA encoding sequence by excellent
Change, i.e., the Preference of codon by its DNA encoding sequence by optimizing, and is synthesized in vitro according to Escherichia coli；
(2), by overlapping pcr, the DNA sequence dna of the self-assembling peptides of synthesis is connected to the DNA encoding sequence of fluorination enzyme
Downstream, i.e. 3 ' ends, be fluorinated enzyme DNA sequence dna be SEQ NO.7, constitute recombinant DNA sequence；
(3) recombinant DNA sequence is inserted into the plasmid of kalamycin resistance, construction recombination plasmid；
Recombinant plasmid import e. coli bl21 (DE3) in, be put into the kanamycins containing 50mg/mL LB culture medium, 37
DEG C culture is until OD600Value is 0.6, then 16 DEG C of inductions, is added 0.05~0.1mM IPTG inducing expression 24 hours, is collected thin
Bacterium；
(5) again by bacterial cell disruption, precipitating is collected in centrifugation, and after being eluted three times to sediment using buffer solution, impurity is removed
It goes, obtains target protein, as fluorination enzyme aggregate.
The fluorination enzyme aggregate of self-assembled short peptide label label as described above is in the biotransformation catalyst side of fluoride
Application in face.
The fluorination enzyme aggregate of self-assembled short peptide label label as described above is preparing positron emission computerized tomography
Application in radioactive tracer.
The advantages of present invention obtains and good effect are as follows:
1, present invention fluorination enzyme aggregate is a kind of fluorination enzyme of Nano grade using self-assembled short peptide label, can
Catalytic efficiency is improved, thermal stability is enhanced and there is reusability, which can apply in fluoride
It neutralizes and is applied in the radioactive tracer for preparing positron emission computerized tomography in terms of biotransformation catalyst.
2, the method for the method of the present invention application genetic engineering (genetic engineering) carries out solvable fluorination enzyme
Molecular modification, using three kinds of self-assembled short peptide labels, building can be improved the catalytic efficiency of enzyme, enhances thermal stability and makes it
Nanoscale with reusability is fluorinated enzyme.Three kinds of plasmids are obtained by gene cloning mode, three kinds of plasmids are converted respectively
Fluorination enzyme aggregate is obtained by inducing expression and purifying into the efficient heterogenous expression of escherichia coli prokaryotic expression system.
3, the observation that the present invention passes through transmission electron microscope, it was demonstrated that three kinds of prepared fluorination enzyme aggregates all form nanoscale
Other protein particulate.
4, enzyme kinetics parameters that the present invention passes through three kinds of fluorination enzyme aggregates of Syrups by HPLC, it was demonstrated that three kinds
Fluorination enzyme aggregate can be catalyzed s-adenosyl-L-methionine (SAM), generate 5 '-fluorinations desoxyadenossine (5 '-FDA).Also,
Experiment also confirms that the FIA-L6KD in three kinds of fluorination enzyme aggregates has higher catalytic efficiency compared to solvable fluorination enzyme,
Stronger thermal stability, and can reuse.Meanwhile according to the nucleoside hydrolase of building, two are carried out together with fluorination enzyme
Enzymatic reaction is walked, illustrates to have using isotope labelling18F synthesis 5 '-18FDR radioactive tracer (radiotracer)
Potentiality, among positron emission computerized tomography (positron emission tomography, PET).
Using Value linear (18F) the fluorine ion marked produces 5 '-by being fluorinated the catalysis of enzyme18FDA, then by some enzymes
(such as: nucleoside hydrolase) catalysis generates the radioactive tracer 5 '-for having same effect with fluorination ribose18FDR(5’-
fluorodeoxyribose,5'-FDR).Using the method for this biosynthesis, the life of radioactive tracer can not only be reduced
Produce cost, and more terre verte environmental protection.So carrying out molecular modification using three kinds of self-assembled short peptide labels to fluorination enzyme, obtaining
The more excellent fluorination enzyme of zymologic property is obtained, also there is very positive meaning to the application and popularization of PET.
5, the method for the present invention prepares nanoscale and is fluorinated enzyme aggregate, to improve enzyme by the transformation to enzyme molecule level
Catalytic efficiency, thermal stability and make it have reusability；This method is using self-assembled short peptide label, and building can mention
The catalytic efficiency of high enzyme, thermal stability and the nanoscale fluorination enzyme aggregate for making it have reusability, it is provided by the invention
Three kinds of fluorination enzyme aggregates (FIA-ELK16, FIA-L6KD, FIA-18A) can be catalyzed S- gland in the presence of inorganic fluorion
Glycosides-l-methionine (SAM) generates 5 '-fluorinations desoxyadenossine (5 '-FDA).
6, nanoscale fluorination enzyme aggregate of the present invention can be prepared into biocatalyst, and the biology applied to fluoride turns
Change；Nanoscale fluorination enzyme aggregate can also be prepared for positron emission computerized tomography (positron emission
Tomography, PET) radioactive tracer, use scope is extensive.
Detailed description of the invention
Fig. 1 is fluorination enzyme aggregate FIA-ELK16, FIA-L6KD, FIA-18A of three kinds of self-assembling peptides label in the present invention
The enzyme reaction schematic diagram that can be catalyzed；It can be seen that fluorination enzyme aggregate can utilize inorganic fluorion (F-), it is catalyzed S- adenosine-
L-methionine (SAM) generates 5 '-fluorinations desoxyadenossine (5 '-FDA) and L-Methionine；
Fig. 2 is that transmission electron microscope observing three kinds of fluorination enzyme aggregates FIA-ELK16, FIA-L6KD, FIA- are used in the present invention
The result figure of the particle size of 18A；Show the particle size of three kinds of fluorination enzyme aggregates in Nano grade in figure；
Fig. 3 is in the present invention using the result figure of HPLC/LC-MS detection fluorination enzyme aggregate reaction product；Wherein liquid phase
Listed enzyme reaction product 5'- is fluorinated desoxyadenossine (5 '-FDA) in chromatogram and mass spectrogram corresponding diagram 1, and arrow meaning is corresponding
Enzyme reaction product liquid chromatogram signal, the structure and its molecular weight of enzyme reaction product be attached on mass spectrogram；
Fig. 4 is the purification result figure of three kinds of fluorinations enzyme aggregate FIA-ELK16, FIA-L6KD, FIA-18A in the present invention；
The wherein purifying that FIA-ELK16 and FIA-L6KD can have, FIA-18A can be also purified；
Fig. 5 is three kinds of fluorination enzyme aggregate FIA-ELK16, FIA-L6KD, FIA-18A in the present invention with S- adenosine-L- first
The rice Mans enzyme kinetics matched curve figure that methyllanthionine (SAM) is obtained as substrate；
Fig. 6 is that there is repetition to make by verifying three kinds of fluorinations enzyme aggregate FIA-ELK16, FIA-L6KD, FIA-18A in the present invention
With the result figure of property；
Fig. 7 is three kinds of fluorination enzyme aggregates and nucleoside hydrolase (TvNH) two step enzymatic reaction result figure in the present invention；Its
In, Fig. 7 A is in the present invention using the result figure of the product adenine (AD) of two step enzymatic reaction of high performance liquid chromatography detection；Figure
7B is the result figure for detecting the molecular weight of adenine in the present invention using LC-MS instrument；Fig. 7 C is that two step enzymatics are anti-in the present invention
The relative productivity result figure of the 5 '-FDR answered.
Specific embodiment
The embodiment of the present invention is described in detail below, it should be noted that the present embodiment is narrative, is not limited
, this does not limit the scope of protection of the present invention.
Raw material used in the present invention is unless otherwise specified conventional commercial product；Used in the present invention
Method is unless otherwise specified the conventional method of this field.
A kind of fluorination enzyme aggregate of self-assembled short peptide label label, the fluorination enzyme aggregate is to pass through self-assembled short peptide
Label and fluorination enzyme are combined and are prepared.
More preferably, the fluorination enzyme aggregate is FIA-ELK16, and the gene order of encoding gene is SEQ NO.1,
The amino acid sequence of encoding gene is SEQ NO.2；
Alternatively, the fluorination enzyme aggregate is FIA-L6KD, the gene order of encoding gene is SEQ NO.3, coding
The amino acid sequence of gene is SEQ NO.4；
Alternatively, the fluorination enzyme aggregate is FIA-18A, the gene order of encoding gene is SEQ NO.5, coding
The amino acid sequence of gene is SEQ NO.6.
More preferably, the optimum temperature of described FIA-ELK16, FIA-L6KD, FIA-18A are respectively 40 DEG C, 50 DEG C, 60 DEG C,
Optimum pH is 6.0.
More preferably, the size of the FIA-ELK16 is 500-600nm；The size of the FIA-L6KD and FIA-18A is
200-300nm。
More preferably, the substrate of the fluorination enzyme aggregate is s-adenosyl-L-methionine, i.e. SAM.
More preferably, the catalysate of the fluorination enzyme aggregate is 5 '-fluorination desoxyadenossines, i.e. 5 '-FDA.
More preferably, the self-assembled short peptide label is ELK16, L6KD or 18A.
The preparation method of the fluorination enzyme aggregate of self-assembled short peptide label label as described above, steps are as follows:
(1) by consulting literatures, the amino acid sequence of self-assembled short peptide label is obtained, by its DNA encoding sequence by excellent
Change, i.e., the Preference of codon by its DNA encoding sequence by optimizing, and is synthesized in vitro according to Escherichia coli；
(2), by overlapping pcr, the DNA sequence dna of the self-assembling peptides of synthesis is connected to the DNA encoding sequence of fluorination enzyme
Downstream, i.e. 3 ' ends, be fluorinated enzyme DNA sequence dna be SEQ NO.7, constitute recombinant DNA sequence；
(3) recombinant DNA sequence is inserted into the plasmid of kalamycin resistance, construction recombination plasmid；
Recombinant plasmid import e. coli bl21 (DE3) in, be put into the kanamycins containing 50mg/mL LB culture medium, 37
DEG C culture until OD value be 0.6, then 16 DEG C induction, be added 0.05~0.1mM IPTG inducing expression 24 hours, collect bacterium；
(5) again by bacterial cell disruption, precipitating is collected in centrifugation, and after being eluted three times to sediment using buffer solution, impurity is removed
It goes, obtains target protein, as fluorination enzyme aggregate.
The fluorination enzyme aggregate of self-assembled short peptide label label as described above can apply the bioconversion in fluoride
In in terms of catalyst.
The fluorination enzyme aggregate of self-assembled short peptide label label as described above can be applied disconnected in preparation positron emission
In the radioactive tracer of layer scanning.
Specifically, the preparation method of the fluorination enzyme aggregate of self-assembled short peptide label label as described above is specific to prepare
Process is as follows:
(1) by consulting literatures, the amino acid sequence of three kinds of self-assembled short peptide label Es LK16, L6KD and 18A are obtained, it will
Its DNA encoding sequence is by optimization, i.e., according to Escherichia coli to the Preference of codon, by its DNA encoding sequence by optimizing,
And it synthesizes in vitro.
(2) by overlapping pcr, the DNA sequence dna of three kinds of self-assembling peptides of synthesis is connected respectively to the DNA of fluorination enzyme
The downstream of coded sequence, i.e. 3 ' ends, the DNA sequence dna for being fluorinated enzyme is SEQ NO.7, constitutes recombinant DNA sequence.
(3) recombinant DNA sequence is inserted into the plasmid of kalamycin resistance, construction recombination plasmid.
(4) recombinant plasmid import e. coli bl21 (DE3) in, be put into the kanamycins containing 50mg/mL LB culture medium, 37
DEG C culture is until OD600Value is 0.6, then 16 DEG C of inductions, is added 0.05~0.1mM IPTG inducing expression 24 hours, is collected thin
Bacterium.
(5) again by bacterial cell disruption, precipitating is collected in centrifugation, after being eluted three times to sediment using buffer solution, by impurity
It removes, obtains target protein, as fluorination enzyme aggregate, as a result as shown in figure 4, showing solvable fluorination enzyme and three kinds of fluorine in figure
Changing enzyme aggregate can express and be purified.
(6) it is fluorinated the research of enzyme aggregate size: utilizing the purified fluorination enzyme aggregate of transmission electron microscope observing, knot
Fruit is as shown in Fig. 2, available three kinds of fluorinations enzyme aggregate all has nano-grade size.
(7) zymologic property research: enzymology is carried out to three kinds of fluorination enzyme aggregates, it is found that it exists in KF (potassium fluoride)
Under, it can be catalyzed s-adenosyl-L-methionine (SAM), generate 5 '-fluorinations desoxyadenossine (5 '-FDA) and L-Methionine, as a result
As shown in Figure 1.Using three kinds of Syrups by HPLC be fluorinated enzyme aggregate zymetology aerodynamic parameter, as a result as shown in figure 3,
Three kinds of fluorination enzyme aggregates are demonstrated compared with solvable fluorination enzyme, wherein the catalytic efficiency of FIA-L6KD is higher.
(8) by carrying out thermal stability and repeatability experiment to three kinds of fluorination enzyme aggregates and solvable fluorination enzyme, as a result
As shown in Figure 6, it was demonstrated that FIA-L6KD has stronger thermal stability in three kinds of fluorination enzyme aggregates；And three kinds of fluorination enzyme aggregations
Body may be reused, and still have 50% or more catalytic activity within nine times.
Finally, carrying out two step enzymatic reactions using constructed nucleoside hydrolase and fluorination enzyme fluorination enzyme aggregate, disclose
Have and utilizes isotope labelling18F synthesis 5 '-18The potentiality of FDR radioactive tracer.
More specifically operating process is as follows:
1. the measurement of three kinds of fluorination enzyme aggregate sequences
The gene order of three kinds of self-assembling peptides is connected respectively to fluorination enzyme sequence downstream, is obtained by overlapping pcr
Gene as described in sequence 1,3,5, three kinds of fluorination enzyme aggregate amino acid sequences are as follows:
FIA-ELK16:
MSADPTQRPIIGFMSDLGTTDDSVAQCKGLMHSICPGVTVIDVCHSMTPWDVEEGARYIVDLPRFFPEG
TVFATTTYPATGTETRSVAVRIKQAAKGGARGQWAGSAGGFERAEGSYIYVAPNNGLLTTVLEEHGYIEAYEVSSTK
VIPERPEPTFYSREMVAIPAAHLAAGFPLSEVGRPLEDSEIVRYQPPQVEISGDTLTGVVSAIDHPYGNVWTNIHRT
HLEKAGIGYGKRIKIILDDVLPFEQTLVPTFADAGEIGGVAAYLNSRGYLSLARNLASLAYPFNLKAGLKVRVETNP
TPPTTPTPPTTPTPTPLELELKLKLELELKLK
FIA-L6KD:
MSADPTQRPIIGFMSDLGTTDDSVAQCKGLMHSICPGVTVIDVCHSMTPWDVEEGARYIVDLPRFFPEG
TVFATTTYPATGTETRSVAVRIKQAAKGGARGQWAGSAGGFERAEGSYIYVAPNNGLLTTVLEEHGYIEAYEVSSTK
VIPERPEPTFYSREMVAIPAAHLAAGFPLSEVGRPLEDSEIVRYQPPQVEISGDTLTGVVSAIDHPYGNVWTNIHRT
HLEKAGIGYGKRIKIILDDVLPFEQTLVPTFADAGEIGGVAAYLNSRGYLSLARNLASLAYPFNLKAGLKVRVETNP
TPPTTPTPPTTPTPTPLLLLLLKD
FIA-18A:
MSADPTQRPIIGFMSDLGTTDDSVAQCKGLMHSICPGVTVIDVCHSMTPWDVEEGARYIVDLPRFFPE
GTVFATTTYPATGTETRSVAVRIKQAAKGGARGQWAGSAGGFERAEGSYIYVAPNNGLLTTVLEEHGYIEAYEVSS
TKVIPERPEPTFYSREMVAIPAAHLAAGFPLSEVGRPLEDSEIVRYQPPQVEISGDTLTGVVSAIDHPYGNVWTNI
HRTHLEKAGIGYGKRIKIILDDVLPFEQTLVPTFADAGEIGGVAAYLNSRGYLSLARNLASLAYPFNLKAGLKVRV
ETNPTPPTTPTPPTTPTPTPEWLKAFYEKVLEKLKELF。
2. the plasmid construction of three kinds of fluorination enzyme aggregates is overexpressed and purifies
Gene is inserted into plasmid vector by double digestion by the DNA encoding sequence that obtain three kinds are fluorinated enzyme aggregate
On, construction recombination plasmid.
Recombinant plasmid application heat shock procedures are transformed into e. coli bl21 (DE3).Specially three kinds of fluorination enzymes are assembled
The plasmid of body and Escherichia coli conversion state cell are uniformly mixed respectively, and 42 DEG C of heating are converted for 90 seconds.There are three types of containing respectively
BL21 (DE3) cell of fluorination enzyme aggregate plasmid is put into the LB culture medium of the kanamycins containing 50mg/mL until in 600nm
(OD600) absorbance value reach 0.6 or so.It is cooled to room temperature, the isopropyl-β-D- sulphur of final concentration of 0.05mM is added
For galactoside (isopropylthiogalactoside, IPTG), 16 DEG C are cultivated 24 hours.
Then cell is collected, cracking, centrifugation, collects precipitating, cleaned three times with lysis buffer, obtain three kinds of aggregations
Body.The albumen then purified is analyzed with polyacrylamide gel electrophoresis (SDS-PAGE), then to be fluorinated enzyme as control, is led to
It is quantitative to aggregation to cross gray analysis software imageJ.
3. efficient liquid phase instrument (HPLC) and LC-MS instrument (LC-MS) detect enzyme reaction product
HPLC/LC-MS is used to the enzyme reaction product that three kinds of fluorination enzyme aggregate catalysis substrates of detection generate.The reaction exists
It is carried out in the phosphate buffer of 20mM, pH 7.8.It include fluorination enzyme aggregate (0.5mg/mL), KF (200mM) and substrate
SAM (200 μM) reacts 30 minutes.After reaction, it is anti-to terminate that the trifluoroacetic acid that mass concentration is 10% is added into system
It answers, then is centrifuged (12000rpm, 10min) except deproteinized, supernatant is for analyzing.
4. the structural form and size of three kinds of fluorination enzyme aggregates of measurement
Three kinds of fluorination enzyme aggregates are observed by saturating color Electronic Speculum (TEM), to determine aggregate structure state and ruler
Very little size.The three kinds of fluorination enzyme aggregates and the solvable fluorination enzyme of its control group of the 10 final concentration of 0.5mg/mL of μ L are taken, then, point
Be not pipetted on the copper mesh of carbon coating of glow discharge, and at room temperature (25 DEG C) be incubated for and 60 seconds and blotted with filter paper.Again by net
Lattice are placed in drop phosphotungstic acid (PTA, 2%v/v, pH 7.0) upper 50 second.Excessive PTA is removed, with transmission electron microscope (TEM)
Observe sample.
5. the enzyme kinetics experiment of three kinds of fluorination enzyme aggregates
Enzyme reaction product is detected by high-efficient liquid phase technique, to determine the respective activity and enzyme power of three kinds of fluorination enzyme aggregates
Learn constant.Enzyme reaction is activated by the fluorination enzyme aggregate (final concentration of 0.5mg/mL) being added.Reaction system is phosphate-buffered
Liquid (20mM, pH 7.8), wherein 200mM KF is added, concentration of substrate is 0 μM to 800 μM.Then, it under same substrate, takes not
With the reaction product at time point, detected using efficient liquid phase instrument.
As obtained by detection, the enzyme reaction curve under different concentration of substrate is drawn, to determine the enzyme reaction under various concentration
Initial rate (in terms of the formation speed of 5 '-FDA), make rice Mans enzymatic kinetic curve.It is final to obtain Vmax、Km、KcatAnd spy
Anisotropic these Enzyme kinetic parameters of constant (specificity constant), as a result as shown in Figure 5.
Table 1 is the rice that three kinds of fluorination enzyme aggregates and solvable fluorination enzyme are obtained using s-adenosyl-L-methionine as substrate
Mans enzyme kinetics parameter
6. the thermal stability and reusability of three kinds of fluorination enzyme aggregates of measurement
Pass through the half-life period (t of three kinds of fluorination enzyme aggregates of measurement1/2), to determine its thermal stability at different temperatures.
At different temperatures, three kinds of inclusion body fluorination enzymes are saved into the corresponding time respectively, then carry out enzymatic reaction.Reaction system is phosphorus
Phthalate buffer (20mM, pH 7.8), comprising including fluorination enzyme aggregate (0.5mg/mL), KF (200mM) and substrate SAM
(200 μM) react 30 minutes.Then, reaction product is detected by high performance liquid chromatograph, determines production concentration, draw enzyme activity
Linearity curve, further according to formula t1/2=ln2/kdIt can be calculated the half-life of three kinds of fluorination enzyme aggregates at different temperatures.
Since three kinds of fluorination enzyme aggregates are after enzymatic reaction, itself and reaction product point can be made by high speed centrifugation
From to reach reusable purpose.
Table 2 is the half-life period (t of three kinds of fluorination enzyme aggregates and solvable fluorination enzyme1/2)。
In addition, the 5 '-FDA of reaction product of fluorination enzyme aggregate can be used as substrate, under the action of nucleoside hydrolase, warp
Enzymatic reaction generates 5 '-FDR and other downstream substrates (such as polypeptide, antibody, affine body molecule etc.) carry out bioconjugate crosslinking
(bio-conjugation), have with this and introduce isotope labelling18The potential ability of F.This18F marker can be used as radiation
Tracer (radiotracer) is used for positron emission computerized tomography (positron emission tomography, PET).
7. 5 ' the biosynthesis of-FDR
Three kinds of fluorination enzyme aggregates are respectively placed in its respectively under optimum temperature, according to above-mentioned enzymatic reaction system, are carried out
React 60min, after reaction, by 95 DEG C, 5min to the enzyme-deactivating in system, by centrifugation (13000r/min,
10min), reaction solution is collected.200 μ L are extracted reaction solution, the Trypanosoma vivax of final concentration of 0.5mg/mL is added thereto
Nucleoside hydrolase (TvNH), after adding water to be settled to 400 μ L, 37 DEG C of reaction 60min, by 95 DEG C, 5min goes out to the enzyme in system
It is living, by centrifugation (13000r/min, 10min), collect reaction solution, the detection for HPLC and LC/MS.
SAM generates 5 '-FDA under the catalysis of fluorination enzyme, and then it generates two kinds of reaction products under the catalysis of TvNH
5 '-FDR and adenine (AD), and the ratio of two kinds of products is 1:1.Since 5 '-FDR do not have UV absorption, and AD is in 260nm
There is stronger UV absorption at place, so knowing the production quantity of 5 '-FDR using the content of detection AD.As a result as shown in Figure 7 A,
For the retention time of AD standard items in 6min or so, the reaction product of four kinds of fluorination enzymes is consistent with standard items retention time, illustrates four
Kind fluorination enzyme and TvNH pass through two step enzymatic reactions, can generate 5 '-FDR.In order to have AD in further confirmatory reaction product, pass through
Cross LC-MS detection, as shown in Figure 7 B, [M+H] at this+=136.1, and the relative molecular mass of AD is M=135.1, it is theoretical
It calculates [M+H]+=136.1, it is consistent with testing result.Therefore, illustrate that TvNH can be catalyzed 5 '-FDA and generate 5 '-FDR again.
Meanwhile, the as yield of 5 '-FDRs quantitative to reaction product AD using HPLC, it is to compare with solvable fluorination enzyme FIA,
Four kinds of fluorination enzymes and TvNH pass through two step enzymatic reactions and generate the relative productivity of 5 '-FDR as seen in figure 7 c.It can be obtained from the figure that one
In the fixed time, since the maximum reaction rate of FIA-18A is higher, so its 5 '-FDA generated is most, lead to 5 '-FDR's
Relative productivity is higher；The maximum reaction rate of FIA-L6KD is close with FIA, so the basic phase of the relative productivity of 5 '-FDR of the two
Together.This experiment, SAM can directly be utilized by also showing, by two step enzymatic reactions, directly 5 '-FDR of generation.Demonstrate benefit
With enzyme is fluorinated, can potentially generate [18F]-fluoride, the feasibility applied to PET.
Although disclosing the embodiment of the present invention for the purpose of illustration, it will be appreciated by those skilled in the art that: not
Be detached from the present invention and spirit and scope of the appended claims in, various substitutions, changes and modifications be all it is possible, therefore, this
The range of invention is not limited to the embodiment and attached drawing disclosure of that.
Sequence
The fluorination enzyme aggregate is FIA-ELK16, and the gene order of encoding gene is SEQ NO.1:
atgtctgcggacccgacccagcgcccgatcattggcttcatgtctgacctgggcactaccgacgactc
cgtggcgcagtgcaaaggtctgatgcactctatctgcccgggtgttaccgttatcgacgtttgccacagcatgacc
ccgtgggacgttgaagaaggtgctcgttacatcgttgacctgccgcgcttcttcccggagggcactgttttcgcga
ccaccacctacccggcgaccggtactgaaacccgtagcgttgcggttcgcatcaaacaggcggcgaaaggcggtgc
gcgtggccagtgggcgggttccgcgggtggtttcgaacgtgcggaaggttcttacatctacgttgcaccgaacaac
ggcctgctgaccaccgttctggaggagcacggctacatcgaagcgtacgaagtttcttctaccaaagttatcccgg
aacgtccggaaccgactttctattctcgtgaaatggttgcgatcccggcagcgcacctggcagctggtttcccgct
gtctgaagttggtcgtccgctggaagattctgaaatcgttcgttatcagccgccgcaggtggaaatcagcggtgac
accctgaccggtgttgtttctgcgatcgaccatccgttcggtaacgtttggaccaacatccaccgtacccacctgg
aaaaagcgggtatcggttacggtaaacgtatcaaaatcatcctggacgacgttctgccgtttgagcagaccctggt
tccgaccttcgcggatgctggtgaaattggcggcgtggcagcgtatctgaactctcgtggttacctgtctctggcg
cgtaacgcggcatccctggcgtatccgtttaacctgaaggcgggtctgaaagttcgtgttgaaaccaacccgacac
cacctactacaccgacgccgcctaccacgccaaccccgactcctctggaattggaactgaaactgaaattagaact
tgaattaaaacttaaataa
The amino acid sequence of its encoding gene is SEQ NO.2:
MSADPTQRPIIGFMSDLGTTDDSVAQCKGLMHSICPGVTVIDVCHSMTPWDVEEGARYIVDLPRFFPE
GTVFATTTYPATGTETRSVAVRIKQAAKGGARGQWAGSAGGFERAEGSYIYVAPNNGLLTTVLEEHGYIEAYEVSS
TKVIPERPEPTFYSREMVAIPAAHLAAGFPLSEVGRPLEDSEIVRYQPPQVEISGDTLTGVVSAIDHPYGNVWTNI
HRTHLEKAGIGYGKRIKIILDDVLPFEQTLVPTFADAGEIGGVAAYLNSRGYLSLARNLASLAYPFNLKAGLKVRV
ETNPTPPTTPTPPTTPTPTPLELELKLKLELELKLK；
The fluorination enzyme aggregate is FIA-L6KD, and the gene order of encoding gene is SEQ NO.3:
atgtctgcggacccgacccagcgcccgatcattggcttcatgtctgacctgggcactaccgacgactc
cgtggcgcagtgcaaaggtctgatgcactctatctgcccgggtgttaccgttatcgacgtttgccacagcatgacc
ccgtgggacgttgaagaaggtgctcgttacatcgttgacctgccgcgcttcttcccggagggcactgttttcgcga
ccaccacctacccggcgaccggtactgaaacccgtagcgttgcggttcgcatcaaacaggcggcgaaaggcggtgc
gcgtggccagtgggcgggttccgcgggtggtttcgaacgtgcggaaggttcttacatctacgttgcaccgaacaac
ggcctgctgaccaccgttctggaggagcacggctacatcgaagcgtacgaagtttcttctaccaaagttatcccgg
aacgtccggaaccgactttctattctcgtgaaatggttgcgatcccggcagcgcacctggcagctggtttcccgct
gtctgaagttggtcgtccgctggaagattctgaaatcgttcgttatcagccgccgcaggtggaaatcagcggtgac
accctgaccggtgttgtttctgcgatcgaccatccgttcggtaacgtttggaccaacatccaccgtacccacctgg
aaaaagcgggtatcggttacggtaaacgtatcaaaatcatcctggacgacgttctgccgtttgagcagaccctggt
tccgaccttcgcggatgctggtgaaattggcggcgtggcagcgtatctgaactctcgtggttacctgtctctggcg
cgtaacgcggcatccctggcgtatccgtttaacctgaaggcgggtctgaaagttcgtgttgaaaccaacccgaccc
ctccaaccacacctacaccgcctacgacaccgacgccaacgccgttactgctgttattactgaaagattaa
The amino acid sequence of its encoding gene is SEQ NO.4:
MSADPTQRPIIGFMSDLGTTDDSVAQCKGLMHSICPGVTVIDVCHSMTPWDVEEGARYIVDLPRFFPE
GTVFATTTYPATGTETRSVAVRIKQAAKGGARGQWAGSAGGFERAEGSYIYVAPNNGLLTTVLEEHGYIEAYEVSS
TKVIPERPEPTFYSREMVAIPAAHLAAGFPLSEVGRPLEDSEIVRYQPPQVEISGDTLTGVVSAIDHPYGNVWTNI
HRTHLEKAGIGYGKRIKIILDDVLPFEQTLVPTFADAGEIGGVAAYLNSRGYLSLARNLASLAYPFNLKAGLKVRV
ETNPTPPTTPTPPTTPTPTPLLLLLLKD；
The fluorination enzyme aggregate is FIA-18A, and the gene order of encoding gene is SEQ NO.5:
atgtctgcggacccgacccagcgcccgatcattggcttcatgtctgacctgggcactaccgacgactc
cgtggcgcagtgcaaaggtctgatgcactctatctgcccgggtgttaccgttatcgacgtttgccacagcatgacc
ccgtgggacgttgaagaaggtgctcgttacatcgttgacctgccgcgcttcttcccggagggcactgttttcgcga
ccaccacctacccggcgaccggtactgaaacccgtagcgttgcggttcgcatcaaacaggcggcgaaaggcggtgc
gcgtggccagtgggcgggttccgcgggtggtttcgaacgtgcggaaggttcttacatctacgttgcaccgaacaac
ggcctgctgaccaccgttctggaggagcacggctacatcgaagcgtacgaagtttcttctaccaaagttatcccgg
aacgtccggaaccgactttctattctcgtgaaatggttgcgatcccggcagcgcacctggcagctggtttcccgct
gtctgaagttggtcgtccgctggaagattctgaaatcgttcgttatcagccgccgcaggtggaaatcagcggtgac
accctgaccggtgttgtttctgcgatcgaccatccgttcggtaacgtttggaccaacatccaccgtacccacctgg
aaaaagcgggtatcggttacggtaaacgtatcaaaatcatcctggacgacgttctgccgtttgagcagaccctggt
tccgaccttcgcggatgctggtgaaattggcggcgtggcagcgtatctgaactctcgtggttacctgtctctggcg
cgtaacgcggcatccctggcgtatccgtttaacctgaaggcgggtctgaaagttcgtgttgaaaccaacccaaccc
ctccgacaacaccgacgccaccgaccacgcctacacctacgccggaatggctgaaagcattttatgaaaaagtgct ggaaaaattaaaagaactgttttaa
The amino acid sequence of its encoding gene is SEQ NO.6:
MSADPTQRPIIGFMSDLGTTDDSVAQCKGLMHSICPGVTVIDVCHSMTPWDVEEGARYIVDLPRFFPE
GTVFATTTYPATGTETRSVAVRIKQAAKGGARGQWAGSAGGFERAEGSYIYVAPNNGLLTTVLEEHGYIEAYEVSS
TKVIPERPEPTFYSREMVAIPAAHLAAGFPLSEVGRPLEDSEIVRYQPPQVEISGDTLTGVVSAIDHPYGNVWTNI
HRTHLEKAGIGYGKRIKIILDDVLPFEQTLVPTFADAGEIGGVAAYLNSRGYLSLARNLASLAYPFNLKAGLKVRV
ETNPTPPTTPTPPTTPTPTPEWLKAFYEKVLEKLKELF。
The DNA sequence dna for being fluorinated enzyme is SEQ NO.7:
Atgtctgcggacccgacccagcgcccgatcattggcttcatgtctgacctgggcactaccgacgactc
cgtggcgcagtgcaaaggtctgatgcactctatctgcccgggtgttaccgttatcgacgtttgccacagcatgacc
ccgtgggacgttgaagaaggtgctcgttacatcgttgacctgccgcgcttcttcccggagggcactgttttcgcga
ccaccacctacccggcgaccggtactgaaacccgtagcgttgcggttcgcatcaaacaggcggcgaaaggcggtgc
gcgtggccagtgggcgggttccgcgggtggtttcgaacgtgcggaaggttcttacatctacgttgcaccgaacaac
ggcctgctgaccaccgttctggaggagcacggctacatcgaagcgtacgaagtttcttctaccaaagttatcccgg
aacgtccggaaccgactttctattctcgtgaaatggttgcgatcccggcagcgcacctggcagctggtttcccgct
gtctgaagttggtcgtccgctggaagattctgaaatcgttcgttatcagccgccgcaggtggaaatcagcggtgac
accctgaccggtgttgtttctgcgatcgaccatccgttcggtaacgtttggaccaacatccaccgtacccacctgg
aaaaagcgggtatcggttacggtaaacgtatcaaaatcatcctggacgacgttctgccgtttgagcagaccctggt
tccgaccttcgcggatgctggtgaaattggcggcgtggcagcgtatctgaactctcgtggttacctgtctctggcg
cgtaacgcggcatccctggcgtatccgtttaacctgaaggcgggtctgaaagttcgtgttgaaaccaac。
Sequence table
<110>University Of Science and Technology Of Tianjin
<120>a kind of fluorination enzyme aggregate of self-assembled short peptide label label and application
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 999
<212> DNA/RNA
<213>gene order (Unknown) for the encoding gene that fluorination enzyme aggregate is FIA-ELK16
<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>amino acid sequence (Unknown) for the encoding gene that fluorination enzyme aggregate is FIA-ELK16
<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 order (Unknown) for the encoding gene that fluorination enzyme aggregate is FIA-L6KD
<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>amino acid sequence (Unknown) for the encoding gene that fluorination enzyme aggregate is FIA-L6KD
<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 order (Unknown) for the encoding gene that fluorination enzyme aggregate is FIA-18A
<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>amino acid sequence (Unknown) for the encoding gene that fluorination enzyme aggregate is FIA-18A
<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>it is fluorinated the DNA sequence dna (Unknown) of enzyme
<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 (10)
1. a kind of fluorination enzyme aggregate of self-assembled short peptide label label, it is characterised in that: the fluorination enzyme aggregate is to pass through
Self-assembled short peptide label and fluorination enzyme are combined and are prepared.
2. the fluorination enzyme aggregate of self-assembled short peptide label label according to claim 1, it is characterised in that: the fluorination
Enzyme aggregate is FIA-ELK16, and the gene order of encoding gene is SEQ NO.1, and the amino acid sequence of encoding gene is
SEQ NO.2；
Alternatively, the fluorination enzyme aggregate is FIA-L6KD, the gene order of encoding gene is SEQ NO.3, encoding gene
Amino acid sequence be SEQ NO.4；
Alternatively, the fluorination enzyme aggregate is FIA-18A, the gene order of encoding gene is SEQ NO.5, encoding gene
Amino acid sequence be SEQ NO.6.
3. the fluorination enzyme aggregate of self-assembled short peptide label label according to claim 2, it is characterised in that: the FIA-
The optimum temperature of ELK16, FIA-L6KD, FIA-18A are respectively 40 DEG C, 50 DEG C, 60 DEG C, and optimum pH is 6.0.
4. the fluorination enzyme aggregate of self-assembled short peptide label label according to claim 2, it is characterised in that: the FIA-
The size of ELK16 is 500-600nm；The size of the FIA-L6KD and FIA-18A is 200-300nm.
5. the fluorination enzyme aggregate of self-assembled short peptide label label according to any one of claims 1 to 4, feature exist
In: the substrate of the fluorination enzyme aggregate is s-adenosyl-L-methionine, i.e. SAM.
6. the fluorination enzyme aggregate of self-assembled short peptide label label according to any one of claims 1 to 4, feature exist
In: the catalysate of the fluorination enzyme aggregate is 5 '-fluorination desoxyadenossines, i.e. 5 '-FDA.
7. the fluorination enzyme aggregate of self-assembled short peptide label label according to any one of claims 1 to 4, feature exist
In: the self-assembled short peptide label is ELK16, L6KD or 18A.
8. the preparation method of the fluorination enzyme aggregate of self-assembled short peptide label label as described in any one of claim 1 to 7,
Be characterized in that: steps are as follows:
(1) by consulting literatures, the amino acid sequence of self-assembled short peptide label is obtained, by its DNA encoding sequence by optimizing, i.e.,
According to Escherichia coli to the Preference of codon, by its DNA encoding sequence by optimizing, and synthesize in vitro；
(2), by overlapping pcr, the DNA sequence dna of the self-assembling peptides of synthesis is connected under the DNA encoding sequence of fluorination enzyme
Trip, i.e. 3 ' ends, the DNA sequence dna for being fluorinated enzyme is SEQ NO.7, constitutes recombinant DNA sequence；
(3) recombinant DNA sequence is inserted into the plasmid of kalamycin resistance, construction recombination plasmid；
Recombinant plasmid import e. coli bl21 (DE3) in, be put into the kanamycins containing 50mg/mL LB culture medium, 37 DEG C training
It supports until OD600Value is 0.6, then 16 DEG C of inductions, is added 0.05~0.1mM IPTG inducing expression 24 hours, and bacterium is collected；
(5) again by bacterial cell disruption, precipitating is collected in centrifugation, and after being eluted three times to sediment using buffer solution, impurity is removed,
Target protein is obtained, as fluorination enzyme aggregate.
9. the fluorination enzyme aggregate of self-assembled short peptide label label as described in any one of claim 1 to 7 is in the life of fluoride
Application in terms of object reforming catalyst.
10. the fluorination enzyme aggregate of self-assembled short peptide label label as described in any one of claim 1 to 7 is preparing positive electron
Application in the radioactive tracer of emission computed tomography.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112760303A (en) * | 2021-02-05 | 2021-05-07 | 天津科技大学 | High-stereoselectivity methionine adenosyltransferase and preparation method and application thereof |
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| CN104755502A (en) * | 2012-10-12 | 2015-07-01 | 清华大学 | Production and purification methods of polypeptide |
| CN108570439A (en) * | 2017-03-13 | 2018-09-25 | 浙江京新药业股份有限公司 | The fusion protein of oxidoreducing enzyme, genetic engineering bacterium and its preparation method and application |
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| CN112760303A (en) * | 2021-02-05 | 2021-05-07 | 天津科技大学 | High-stereoselectivity methionine adenosyltransferase and preparation method and application thereof |
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