CN106749602A - It is a kind of to help expressed sequence and its application in acellular expression ADCY2 albumen - Google Patents
It is a kind of to help expressed sequence and its application in acellular expression ADCY2 albumen Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
- C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
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- C12Y—ENZYMES
- C12Y406/00—Phosphorus-oxygen lyases (4.6)
- C12Y406/01—Phosphorus-oxygen lyases (4.6.1)
- C12Y406/01001—Aodenylate cyclase (4.6.1.1)
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- C12N2800/00—Nucleic acids vectors
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- C12N2800/101—Plasmid DNA for bacteria
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- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
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Abstract
Expressed sequence and its application in acellular expression ADCY2 albumen are helped the invention discloses a kind of, belongs to biological technical field.This helps the amino acid sequence of expressed sequence to have the amino acid sequence of at least 75% homology as shown in sequence table SEQ ID1 or with it.This helps expressed sequence to be mainly used in the acellular expression of multiple transmembrane protein.It is of the invention it is a kind of help expressed sequence and its application process in acellular expression ADCY2 albumen, can solve the problems, such as that acellular albumen expression quantity is low.
Description
Technical field
The present invention relates to acellular expression field, and in particular to one kind helps expressed sequence and its in acellular expression ADCY2
Application in albumen.
Background technology
26S Proteasome Structure and Function analysis needs substantial amounts of memebrane protein sample, but a bottleneck is how high-volume is efficiently obtained
Memebrane protein.Cell free expression system is the Transcription/Translation System of coupling, reduces the complexity of expressing protein.Using helping expression sequence
The tactful optimization that can be based on translation initiation of row brings the quick raising of acellular expression efficiency.High frequency uses rare codon
And unfavorable initiation of translation has been confirmed as the main limiting step of Protein synthesis, in the 5' end regions of mRNA, such as core
Sugared body binding site is related to the formation of the secondary structure of excessive risk to slow down or even prevents startup program, the rare codon of high frequency
Son is likely to result in translation and pauses, the amino acid of mispairing, or even premature end event, but such problem can be by synthetic gene table
The optimization that reaches is solved.
Adenyl cyclase, abbreviation ADCY is integral membrane protein, and 12 transmembrane glycoproteins are the effector of G-protein, energy
Catalysis ATP generation cAMP, cause cell response.Adenyl cyclase is distributed widely in the cell membrane of mammal, and this enzyme is urged
Change ATP generation cAMP and discharge pyrophosphoric acid.Adenyl cyclase is distributed mainly on cytoplasma membrane, nuclear membrane and endoplasmic reticulum.Gland
Thuja acid cyclase (ADCY) is the key signal molecule in g protein coupled receptor (GPCRs) downstream.It is of substantially standard acellular to be
Be beyond expression out complete protein molecular, so needing to be improved acellular expression condition.
The content of the invention
For problems of the prior art, expressed sequence is helped and its without thin it is an object of the invention to provide one kind
Application in cellular expression ADCY2 albumen, can solve the problems, such as that acellular albumen expression quantity is low.
The present invention is achieved through the following technical solutions：One kind of the invention helps expressed sequence, the ammonia for helping expressed sequence
Base acid sequence has the amino acid sequence of at least 75% homology as shown in sequence table SEQ ID1 or with it.
Further, the acellular expression for helping expressed sequence to be mainly used in multiple transmembrane protein.
Present invention also offers it is a kind of it is as described above help expressed sequence in acellular expression ADCY2 albumen should
With its application process is as follows：
Step (1)：By ADCY2 GFPs to be expressed carry out after codon optimization with the gene and group for helping expressed sequence
His tag is attached；
Step (2)：Design primer simultaneously introduces double enzyme site, and the purpose fragment that connection in step (1) is obtained is cloned into
On expression vector pET28a containing T7 promoters, expression plasmid is obtained；
Step (3)：Acellular albumen expression system is prepared, the expression system includes following composition：HEPES-KOH is buffered
Liquid, ATP, GTP, UTP, CTP, cAMP, potassium glutamate, ammonium acetate, magnesium acetate, folinic acid, t7 rna polymerase, amino acid,
PET8000, PEP, Escherichia coli extract；The expression plasmid that will be obtained in step (2) adds acellular egg
Expressed in white expression system.
As further preferably, ADCY2 genes carry out the sequence such as sequence table after codon optimization in the step (1)
Shown in 2.
As further preferred, in the step (1), by SOE-PCR methods and flush end connection method in ADCY2
The N-terminal connection of the fragment after gene codon optimization helps the genetic fragment of expressed sequence, C-terminal connection histidine-tagged.
As it is further preferably, in the step (1), ADCY2 genes and help expressed sequence also to contain protease digestion
Site sequence, the protease cleavage site sequence is fibrin ferment restriction enzyme site or enterokinase cleavage site.
It is semicontinuous mass exchange reaction during the expression, by the composition in expression system as further preferred
Be put into dialysis tubing, and by dialysis tubing be placed in containing supply reagent centrifuge tube in expressed.
As further preferably, the supply reagent includes following composition：HEPES-KOH buffer solutions, cAMP, glutamic acid
Potassium, ammonium acetate, magnesium acetate, folinic acid, amino acid, PEP, PET8000, Escherichia coli extract.
Beneficial effects of the present invention are：The present invention helps the expressed sequence to carry out acellular table by the addition on ADCY2 genes
Reach, expressing quantity is high, solve the problems, such as that acellular albumen expression quantity is low.
Brief description of the drawings
Fig. 1 is pET28a-ADCY2 Vector maps after building；
Fig. 2 is the protein purification electrophoretogram after the acellular expression of ADCY2 of embodiment 1；
Fig. 3 is the protein purification electrophoretogram after the acellular expression of the ADCY2 of comparative example 1 and comparative example 2.
Specific embodiment
The present invention is described in further detail with implementation method below in conjunction with the accompanying drawings.Following examples are only exemplary
, only it is used to do technical scheme further describe in detail, it will be understood by those within the art that,
In the spirit and scope without departing from technical solution of the present invention, the modification or replacement carried out to technical scheme all should be covered at this
In the claims of invention.
One kind that the present invention is provided helps expressed sequence, the amino acid sequence such as sequence table SEQ ID1 for helping expressed sequence
Amino acid sequence shown or that there is at least 75% homology with it.It is described to help expressed sequence to be mainly used in multiple cross-film egg
White acellular expression.
The concrete application method that expressed sequence is helped in acellular expression ADCY2 albumen of the invention is as follows：
Step (1)：By ADCY2 GFPs to be expressed carry out after codon optimization with the gene and group for helping expressed sequence
His tag is attached；
Step (2)：Design primer simultaneously introduces double enzyme site, and the purpose fragment that connection in step (1) is obtained is cloned into
On expression vector pET28a containing T7 promoters, expression plasmid is obtained；
Step (3)：Acellular albumen expression system is prepared, the expression system includes following composition：HEPES-KOH is buffered
Liquid, ATP, GTP, UTP, CTP, cAMP, potassium glutamate, ammonium acetate, magnesium acetate, folinic acid, t7 rna polymerase, amino acid,
PET8000, PEP, Escherichia coli extract；The expression plasmid that will be obtained in step (2) adds acellular egg
Expressed in white expression system.
In step (1), ADCY2 genes carry out the sequence after codon optimization as shown in sequence table 2.By SOE-PCR side
The N-terminal connection of the fragment of method and flush end connection method after the optimization of ADCY2 gene codons help expressed sequence genetic fragment,
C-terminal connection is histidine-tagged, and three sections of sequences connect into whole sequence, as the template that PCR reacts, ADCY2 genes and helps expression
Sequence also contains protease cleavage site sequence, and the protease cleavage site sequence is fibrin ferment restriction enzyme site or enterokinase enzyme
Enzyme site；
In step (2), design introduce the restriction enzyme sites of Nco I sense primer (5 '-
ATGCTCCATGGATCCAGCGTACTCCAAAGATT-3 ') and introduce the restriction enzyme sites of Xho I anti-sense primer (5 '-
GGGCCCTCGAGATGATGATGATGATGATGATGATGATGATG-3 '), to connect purpose fragment as template, add DNA to gather
Synthase and 10 times of DNA polymerase buffer liquid carry out pcr amplification reaction, after amplified production is checked through 1% agarose gel electrophoresis,
DNA purifying is carried out, genetic fragment to be expressed after purification carries out double digestion, after being checked through 1.0% agarose gel electrophoresis, DNA
Purification Kit is reclaimed, and the genetic fragment to be expressed containing restriction enzyme site is mixed with linear pET28a plasmids, adds T4DNA
Ligase, 10 × T4DNA ligase buffer solutions, connection liquid conversion competent escherichia coli cell DH5 α, take positive colony bacterial strain
DNA sequencing is carried out, to recognizing through sequencing confirmation, the bacterium colony containing pET28a recombinant plasmids carries out plasmid purification, obtains pET28a-
ADCY2 plasmids.
It is described to be expressed as the reaction of semicontinuous mass exchange in step (3), by expression system include following reaction solution into
Point：57mM HEPES-KOH buffer solutions (pH8.2), 1.2mM ATP, 0.85mM GTP, 0.85mM UTP, 0.85mM CTP,
0.64mM cAMP, 200mM potassium glutamate, 80mM ammonium acetates, 15mM magnesium acetates, 34 μ g/ml folinic acid, 33 μ g/ml T7RNA gather
Synthase, 500 μM of amino acid, 2%PET8000,33mM PEP and 24% Escherichia coli extracts；Take 300 μ l
Reaction solution adds 6.7 μ g/ml pET28a-ADCY2 plasmids in dialysis tubing；Supply reagent includes following composition：57mM
HEPES-KOH buffer solutions, 0.64mM cAMP, 200mM potassium glutamate, 80mM ammonium acetates, 15mM magnesium acetates, 34 μ g/mL Asias leaf
Acid, 500 μM of amino acid, 33mM PEPs, 2%PET8000,24% Escherichia coli extracts；Supply reagent dress
In centrifuge tube；The dialysis tubing that will be equipped with reaction solution is placed in acellular albumen expression is carried out in centrifuge tube, by SDS-PAGE come
Detect the expression of ADCY2.
Embodiment 1
1. pair ADCY2 genes carry out codon optimization, and the Codon sequences after optimization are shown in sequence table 2, in codon optimization
N sections of addition of the ADCY2 genes of sequence afterwards helps expressed sequence B-tag
(ATCCAGCGTACTCCAAAGATTCAGGTTTACTCACGTCATCCAGCAGAGAATGGAAAGTCAAATTTCCTGAATTGCT
AT；Amino acid sequence is IQRTPKIQVYSRHPAENGKSNFLNCY), C-terminal addition 10 × his (serine) label
(CATCATCATCATCATCATCATCATCATCAT；HHHHHHHHHH), the whole sequence of gene chemical synthesis, as the mould of gene cloning
Plate.
2. the template reacted as PCR containing B-tag and 10 × his labels purpose fragment of above-mentioned synthesis is taken, and design is introduced
Sense primer (the 5 '-ATGCT of the restriction enzyme sites of Nco ICCATGGATCCAGCGTACTCCAAAGATT-3 ') and introduce the digestions of Xho I
Anti-sense primer (the 5 '-GGGCC in siteCTCGAGATGATGATGATGATGATGATGATGATGATG-3’).4.0 μ L moulds are taken respectively
Plate, the upstream and downstream primer of 2 μ L, 10 μm of ol/L, 1.0 μ L archaeal dna polymerases (5U/ μ L), 10 μ 10 × DNA polymerase buffers of L
Liquid, 5.0 μ L dNTP mixing are used as PCR reaction systems.Reaction solution is adjusted to 50 μ L, the predegeneration under the conditions of 95 DEG C with sterilized water
2min is processed, 30s, 65 DEG C of annealing 30s, 72 DEG C of extension 30s, final 72 DEG C whole extension 5min are then denatured at 94 DEG C respectively, it is whole
Individual PCR reaction systems carry out 35 circulations altogether.After amplified production is checked through 1% agarose gel electrophoresis, DNA purifying is carried out.It is pure
ADCY2 fragments after change carry out double digestion, and reaction system is as follows：30 μ L with the addition of the ADCY2 fragments of restriction enzyme site, 5 μ L 10 ×
Buffer solution, 1 μ L Nco I (20U/ μ L), l μ L Xho I (20U/ μ L), after reaction solution is adjusted into 50 μ L with sterilized water, at 37 DEG C
Insulation 1h, 20min is then processed under the conditions of 65 DEG C carries out heat inactivation.After being checked through 1.0% agarose gel electrophoresis, DNA is pure
Change kits to reclaim.On the other hand, pET28a plasmid vectors are converted into bacillus coli DH 5 alpha, matter is extracted after overnight incubation
Grain, and double digestion is carried out, after being checked through 1.0% agarose gel electrophoresis, cutting linear plasmid band carries out purifying recovery.
Structure (the pET28a-ADCY2 Vector maps such as Fig. 1 institutes after structure of 3.pET28a-ADCY2 recombinant expression carriers
Show)：The above-mentioned ADCY2 fragments containing restriction enzyme site are pressed 2 with linear pET28a plasmids:1 ratio, be incubated 5min at 45 DEG C after,
It is placed in ice bath and cools down, 1 μ L T4DNA ligases, 2 μ L 10 × T4DNA ligase buffer solutions, l6 DEG C of guarantor is separately added into after cooling
Warm 16h.Connection liquid conversion competent escherichia coli cell DH5 α, through 37 DEG C of overnight incubations after, select 3~5 bacterium colonies and be PCR and test
Card, further carries out digestion identification, and positive colony is used for DNA sequencing.To through sequencing confirmation, containing pET28a-ADCY2 recombinate matter
The bacterium colony of grain carries out plasmid purification.
4. the synthesis of the semicontinuous reaction expression system of acellular albumen, reaction solution includes following composition：57mM HEPES-
KOH buffer solutions (pH8.2), 1.2mM ATP, 0.85mM GTP, 0.85mM UTP, 0.85mM CTP, 0.64mM cAMP, 200mM
Potassium glutamate, 80mM ammonium acetates, 15mM magnesium acetates, 34 μ g/ml folinic acid, 33 μ g/ml t7 rna polymerases, 500 μM of amino acid,
2%PET8000,33mM PEP and 24% Escherichia coli extract；300 μ l reaction solutions are taken in D-TubeTMThoroughly
In analysis pipe, 6.7 μ g/ml pET28a-ADCY2 plasmids are added；4.5mL supply reagents are taken loaded in 50mL centrifuge tubes, reagent is supplied
Including following composition：57mM HEPES-KOH buffer solutions, 0.64mM cAMP, 200mM potassium glutamate, 80mM ammonium acetates, 15mM vinegar
Sour magnesium, 34 μ g/mL folinic acid, 500 μM of amino acid, 33mM PEPs, 2%PET8000,24% Escherichia coli are taken out
Extract；The dialysis tubing that will be equipped with reaction solution is placed in acellular albumen expression is carried out in centrifuge tube, is detected by SDS-PAGE
The expression of ADCY2.
Comparative example 1
1. pair ADCY2 genes carry out codon optimization, and the Codon sequences after optimization are shown in sequence table 2, in codon optimization
The C-terminal of the ADCY2 genes of sequence adds 10 × his labels (CATCATCATCATCATCATCATCATCATCAT afterwards；
HHHHHHHHHH), the whole sequence of gene chemical synthesis, as the template of gene cloning.
2. the template reacted as PCR containing 10 × his labels purpose fragment of above-mentioned synthesis is taken, and design introduces the digestions of Nco I
Sense primer (the 5 '-ATGCTCCATGG ATGTGGCAGGAA in site
GCTATG-3 ') and introduce the restriction enzyme sites of Xho I anti-sense primer (5 '-
GGGCCCTCGAGATGATGATGATGATGATGATGATGATGATG-3’).4.0 μ L templates, 2 μ L 10 μm of ol/L are taken respectively
Upstream and downstream primer, 1.0 μ L archaeal dna polymerases (5U/ μ L), 10 μ 10 × DNA polymerase buffers of L liquid, 5.0 μ L dNTP mixing
As PCR reaction systems.Reaction solution is adjusted to 50 μ L with sterilized water, predegeneration treatment 2min, then distinguishes under the conditions of 95 DEG C
30s, 65 DEG C of annealing 30s, 72 DEG C of extension 30s, final 72 DEG C whole extension 5min are denatured at 94 DEG C, whole PCR reaction systems are entered altogether
35 circulations of row.After amplified production is checked through 1% agarose gel electrophoresis, DNA purifying is carried out.ADCY2 fragments after purification are entered
Row double digestion, reaction system is as follows：30 μ L with the addition of the ADCY2 fragments of restriction enzyme site, 5 10 × buffer solutions of μ L, 1 μ L Nco I
(20U/ μ L), l μ L Xho I (20U/ μ L), after reaction solution is adjusted into 50 μ L with sterilized water, in 1h is incubated at 37 DEG C, then 65
20min is processed under the conditions of DEG C carries out heat inactivation.After being checked through 1.0% agarose gel electrophoresis, DNA Purification Kits are returned
Receive.On the other hand, pET28a plasmid vectors are converted into bacillus coli DH 5 alpha, plasmid are extracted after overnight incubation, and carry out double digestion,
After being checked through 1.0% agarose gel electrophoresis, cutting linear plasmid band carries out purifying recovery.
The structure of 3.pET28a-ADCY2 recombinant expression carriers：By the above-mentioned ADCY2 fragments containing restriction enzyme site with it is linear
PET28a plasmids press 2:1 ratio, be incubated 5min at 45 DEG C after, be placed in ice bath and cool down, 1 μ L T4DNA are separately added into after cooling
Ligase, 2 μ L 10 × T4DNA ligase buffer solutions, l6 DEG C of insulation 16h.Connection liquid conversion competent escherichia coli cell DH5
α, through 37 DEG C of overnight incubations after, select 3~5 bacterium colonies and do PCR checkings, further carry out digestion identification, positive colony is used for DNA
Sequencing.Plasmid purification is carried out to confirmed through the sequencing, bacterium colony containing pET28a-ADCY2 recombinant plasmids.
4. the synthesis of the semicontinuous reaction expression system of acellular albumen, reaction solution includes following composition：57mM HEPES-
KOH buffer solutions (pH8.2), 1.2mM ATP, 0.85mM GTP, 0.85mM UTP, 0.85mM CTP, 0.64mM cAMP, 200mM
Potassium glutamate, 80mM ammonium acetates, 15mM magnesium acetates, 34 μ g/ml folinic acid, 33 μ g/ml t7 rna polymerases, 500 μM of amino acid,
2%PET8000,33mM PEP and 24% Escherichia coli extract.300 μ l reaction solutions are taken in D-TubeTMThoroughly
In analysis pipe (3.5kDa), 6.7 μ g/ml pET28a-ADCY2 plasmids are added；4.5mL supply reagents are taken to be loaded in 50mL centrifuge tubes,
Supply reagent includes following composition：57mM HEPES-KOH buffer solutions, 0.64mM cAMP, 200mM potassium glutamate, 80mM acetic acid
Ammonium, 15mM magnesium acetates, 34 μ g/mL folinic acid, 500 μM of amino acid, 33mM PEPs, 2%PET8000,24%
Escherichia coli extract；The dialysis tubing that will be equipped with reaction solution is placed in acellular albumen expression is carried out in centrifuge tube, by SDS-
PAGE detects the expression of ADCY2.
Comparative example 2
1. the template that the cloned sequence containing ADCY2 genes reacts as PCR is taken, and design introduces the upstream of the restriction enzyme sites of Nco I
Primer (5 '-ATGCTCCATGGATGTGGCAGGAGGCG-3 ') and introduce the restriction enzyme sites of Xho I anti-sense primer (5 '-
GGGCCCTCGAGGGATGCCACGTTGCTCTGGG-3’).4.0 μ L templates, 2 μ L, 10 μm of upstream and downstreams of ol/L are taken respectively
Primer, 1.0 μ L archaeal dna polymerases (5U/ μ L), 10 μ 10 × DNA polymerase buffers of L liquid, 5.0 μ L dNTP mixing are anti-as PCR
Answer system.Reaction solution is adjusted to 50 μ L with sterilized water, the predegeneration treatment 2min under the conditions of 95 DEG C, then respectively in 94 DEG C of denaturation
30s, 65 DEG C of annealing 30s, 72 DEG C of extension 30s, final 72 DEG C whole extension 5min, whole PCR reaction systems carry out 35 circulations altogether.
After amplified production is checked through 1% agarose gel electrophoresis, DNA purifying is carried out.ADCY2 fragments after purification carry out double digestion, instead
Answer system as follows：30 μ L with the addition of the ADCY2 fragments of restriction enzyme site, 5 10 × buffer solutions of μ L, 1 μ L Nco I (20U/ μ L), l μ L
Xho I (20U/ μ L), after reaction solution is adjusted into 50 μ L with sterilized water, in 1h is incubated at 37 DEG C, is then processed under the conditions of 65 DEG C
20min carries out heat inactivation.After being checked through 1.0% agarose gel electrophoresis, DNA Purification Kits are reclaimed.On the other hand, will
PET28a plasmid vectors convert bacillus coli DH 5 alpha, and plasmid is extracted after overnight incubation, and carry out double digestion, through 1.0% agarose
After gel electrophoresis inspection, cutting linear plasmid band carries out purifying recovery.
The structure of 2.pET28a-ADCY2 recombinant expression carriers：By the above-mentioned ADCY2 fragments containing restriction enzyme site with it is linear
PET28a plasmids press 2:1 ratio, be incubated 5min at 45 DEG C after, be placed in ice bath and cool down, 1 μ L T4DNA are separately added into after cooling
Ligase, 2 μ L 10 × T4DNA ligase buffer solutions, l6 DEG C of insulation 16h.Connection liquid conversion competent escherichia coli cell DH5
α, through 37 DEG C of overnight incubations after, select 3~5 bacterium colonies and do PCR checkings, further carry out digestion identification, positive colony is used for DNA
Sequencing.Plasmid purification is carried out to confirmed through the sequencing, bacterium colony containing pET28a-ADCY2 recombinant plasmids.
3. the synthesis of the semicontinuous reaction expression system of acellular albumen, reaction solution includes following composition：57mM HEPES-
KOH buffer solutions (pH8.2), 1.2mM ATP, 0.85mM GTP, 0.85mM UTP, 0.85mM CTP, 0.64mM cAMP, 200mM
Potassium glutamate, 80mM ammonium acetates, 15mM magnesium acetates, 34 μ g/ml folinic acid, 33 μ g/ml t7 rna polymerases, 500 μM of amino acid,
2%PET8000,33mM PEP and 24% Escherichia coli extract；300 μ l reaction solutions are taken in D-TubeTMThoroughly
In analysis pipe (3.5kDa), 6.7 μ g/ml pET28a-ADCY2 plasmids are added；4.5mL supply reagents are taken to be loaded in 50mL centrifuge tubes,
Supply reagent includes following composition：57mM HEPES-KOH buffer solutions, 0.64mM cAMP, 200mM potassium glutamate, 80mM acetic acid
Ammonium, 15mM magnesium acetates, 34 μ g/mL folinic acid, 500 μM of amino acid, 33mM PEPs, 2%PET8000,24%
Escherichia coli extract；The dialysis tubing that will be equipped with reaction solution is placed in acellular albumen expression is carried out in centrifuge tube, by SDS-
PAGE detects the expression of ADCY2.
Protein purification electrophoretogram after the acellular expression of ADCY2 of embodiment 1 is as shown in Fig. 2 comparative example 1 and comparative example 2
The acellular expression of ADCY2 after protein purification electrophoretogram it is as shown in Figure 3.It is of the invention by adding from Fig. 2 and Fig. 3
The acellular expression of ADCY2 is obvious after helping expressed sequence, and expression quantity is high.
<110>Wuhan Sino-American Biotechnology Company
China, Quan Gao
<120>It is a kind of to help expressed sequence and its application in acellular expression ADCY2 albumen
<130> 1
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 26
<212> PRT
<213> Homo sapiens
<400> 1
Ile Gln Arg Thr Pro Lys Ile Gln Val Tyr Ser Arg His Pro Ala Glu
1 5 10 15
Asn Gly Lys Ser Asn Phe Leu Asn Cys Tyr
20 25
<210> 2
<211> 3273
<212> DNA
<213> Homo sapiens
<400> 2
atgtggcagg aagctatgcg tcgtcgtcgt tacctgcgtg accgttctga agaagctgct 60
ggtggtggtg acggtctgcc gcgttctcgt gactggctgt acgaatctta ctactgcatg 120
tctcagcagc acccgctgat cgttttcctg ctgctgatcg ttatgggttc ttgcctggct 180
ctgctggctg ttttcttcgc tctgggtctg gaagttgaag accacgttgc tttcctgatc 240
accgttccga ccgctctggc tatcttcttc gctatcttca tcctggtttg catcgaatct 300
gttttcaaaa aactgctgcg tctgttctct ctggttatct ggatctgcct ggttgctatg 360
ggttacctgt tcatgtgctt cggtggtacc gtttctccgt gggaccaggt ttctttcttc 420
ctgttcatca tcttcgttgt ttacaccatg ctgccgttca acatgcgtga cgctatcatc 480
gcttctgttc tgacctcttc ttctcacacc atcgttctgt ctgtttgcct gtctgctacc 540
ccgggtggta aagaacacct ggtttggcag atcctggcta acgttatcat cttcatctgc 600
ggtaacctgg ctggtgctta ccacaaacac ctgatggaac tggctctgca gcagacctac 660
caggacacct gcaactgcat caaatctcgt atcaaactgg aattcgaaaa acgtcagcag 720
gaacgtctgc tgctgtctct gctgccggct cacatcgcta tggaaatgaa agctgaaatc 780
atccagcgtc tgcagggtcc gaaagctggt cagatggaaa acaccaacaa cttccacaac 840
ctgtacgtta aacgtcacac caacgtttct atcctgtacg ctgacatcgt tggtttcacc 900
cgtctggctt ctgactgctc tccgggtgaa ctggttcaca tgctgaacga actgttcggt 960
aaattcgacc agatcgctaa agaaaacgaa tgcatgcgta tcaaaatcct gggtgactgc 1020
tactactgcg tttctggtct gccgatctct ctgccgaacc acgctaaaaa ctgcgttaaa 1080
atgggtctgg acatgtgcga agctatcaaa aaagttcgtg acgctaccgg tgttgacatc 1140
aacatgcgtg ttggtgttca ctctggtaac gttctgtgcg gtgttatcgg tctgcagaaa 1200
tggcagtacg acgtttggtc tcacgacgtt accctggcta accacatgga agctggtggt 1260
gttccgggtc gtgttcacat ctcttctgtt accctggaac acctgaacgg tgcttacaaa 1320
gttgaagaag gtgacggtga catccgtgac ccgtacctga aacagcacct ggttaaaacc 1380
tacttcgtta tcaacccgaa aggtgaacgt cgttctccgc agcacctgtt ccgtccgcgt 1440
cacaccctgg acggtgctaa aatgcgtgct tctgttcgta tgacccgtta cctggaatct 1500
tggggtgctg ctaaaccgtt cgctcacctg caccaccgtg actctatgac caccgaaaac 1560
ggtaaaatct ctaccaccga cgttccgatg ggtcagcaca acttccagaa ccgtaccctg 1620
cgtaccaaat ctcagaaaaa acgtttcgaa gaagaactga acgaacgtat gatccaggct 1680
atcgacggta tcaacgctca gaaacagtgg ctgaaatctg aagacatcca gcgtatctct 1740
ctgctgttct acaacaaagt tctggaaaaa gaataccgtg ctaccgctct gccggctttc 1800
aaatactacg ttacctgcgc ttgcctgatc ttcttctgca tcttcatcgt tcagatcctg 1860
gttctgccga aaacctctgt tctgggtatc tctttcggtg ctgctttcct gctgctggct 1920
ttcatcctgt tcgtttgctt cgctggtcag ctgctgcagt gctctaaaaa agcttctccg 1980
ctgctgatgt ggctgctgaa atcttctggt atcatcgcta accgtccgtg gccgcgtatc 2040
tctctgacca tcatcaccac cgctatcatc ctgatgatgg ctgttttcaa catgttcttc 2100
ctgtctgact ctgaagaaac catcccgccg accgctaaca ccaccaacac ctctttctct 2160
gcttctaaca accaggttgc tatcctgcgt gctcagaacc tgttcttcct gccgtacttc 2220
atctactctt gcatcctggg tctgatctct tgctctgttt tcctgcgtgt taactacgaa 2280
ctgaaaatgc tgatcatgat ggttgctctg gttggttaca acaccatcct gctgcacacc 2340
cacgctcacg ttctgggtga ctactctcag gttctgttcg aacgtccggg tatctggaaa 2400
gacctgaaaa ctatgggttc tgtttctctg tctatcttct tcatcaccct gctggttctg 2460
ggtcgtcaga acgaatacta ctgccgtctg gacttcctgt ggaaaaacaa attcaaaaaa 2520
gaacgtgaag aaatcgaaac tatggaaaac ctgaaccgtg ttctgctgga aaacgttctg 2580
ccggctcacg ttgctgaaca cttcctggct cgttctctga aaaacgaaga actgtaccac 2640
cagtcttacg actgcgtttg cgttatgttc gcttctatcc cggacttcaa agaattctac 2700
accgaatctg acgttaacaa agaaggtctg gaatgcctgc gtctgctgaa cgaaatcatc 2760
gctgacttcg acgacctgct gtctaaaccg aaattctctg gtgttgaaaa aatcaaaacc 2820
atcggttcta cctacatggc tgctaccggt ctgtctgctg ttccgtctca ggaacactct 2880
caggaaccgg aacgtcagta catgcacatc ggtactatgg ttgaattcgc tttcgctctg 2940
gttggtaaac tggacgctat caacaaacac tctttcaacg acttcaaact gcgtgttggt 3000
atcaaccacg gtccggttat cgctggtgtt atcggtgctc agaaaccgca gtacgacatc 3060
tggggtaaca ccgttaacgt tgcttctcgt atggactcta ccggtgttct ggacaaaatc 3120
caggttaccg aagaaacctc tctggttctg cagaccctgg gttacacctg cacctgccgt 3180
ggtatcatca acgttaaagg taaaggtgac ctgaaaacct acttcgttaa caccgaaatg 3240
tctcgttctc tgtctcagtc taacgttgct tct 3273
Claims (8)
1. one kind helps expressed sequence, it is characterised in that：The amino acid sequence for helping expressed sequence is as shown in sequence table SEQ ID1
Or there is the amino acid sequence of at least 75% homology with it.
2. expressed sequence is helped as claimed in claim 1, it is characterised in that：It is described to help expressed sequence to be mainly used in multiple cross-film
The acellular expression of albumen.
3. it is a kind of to help application of the expressed sequence in acellular expression ADCY2 albumen, its feature as described in claim 1~2
It is：Its application process is as follows：
Step (1)：By ADCY2 GFPs to be expressed carry out after codon optimization with the gene and histidine for helping expressed sequence
Label is attached；
Step (2)：Design primer simultaneously introduces double enzyme site, and the purpose fragment that connection in step (1) is obtained is cloned into containing T7
On the expression vector pET28a of promoter, expression plasmid is obtained；
Step (3)：Acellular albumen expression system is prepared, the expression system includes following composition：HEPES-KOH buffer solutions,
ATP, GTP, UTP, CTP, cAMP, potassium glutamate, ammonium acetate, magnesium acetate, folinic acid, t7 rna polymerase, amino acid,
PET8000, PEP, Escherichia coli extract；The expression plasmid that will be obtained in step (2) adds acellular egg
Expressed in white expression system.
4. application of the expressed sequence in acellular expression ADCY2 albumen is helped as claimed in claim 3, it is characterised in that：Institute
Stating ADCY2 genes in step (1) carries out the sequence after codon optimization as shown in sequence table 2.
5. application of the expressed sequence in acellular expression ADCY2 albumen is helped as claimed in claim 4, it is characterised in that：It is logical
The N-terminal connection for crossing the fragment of SOE-PCR methods and flush end connection method after the optimization of ADCY2 gene codons helps expressed sequence
Genetic fragment, C-terminal connection it is histidine-tagged.
6. application of the expressed sequence in acellular expression ADCY2 albumen is helped as claimed in claim 5, it is characterised in that：Institute
State in step (1) ADCY2 genes and help expressed sequence also to contain protease cleavage site sequence, the protease cleavage site sequence
It is classified as fibrin ferment restriction enzyme site or enterokinase cleavage site.
7. application of the expressed sequence in acellular expression ADCY2 albumen is helped as claimed in claim 6, it is characterised in that：Institute
State and be expressed as the reaction of semicontinuous mass exchange, the composition in expression system is put into dialysis tubing, and dialysis tubing is placed in contains
Have supply reagent centrifuge tube in expressed.
8. application of the expressed sequence in acellular expression ADCY2 albumen is helped as claimed in claim 7, it is characterised in that：Institute
Stating supply reagent includes following composition：HEPES-KOH buffer solutions, cAMP, potassium glutamate, ammonium acetate, magnesium acetate, folinic acid, ammonia
Base acid, PEP, PET8000, Escherichia coli extract.
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| CN111019963A (en) * | 2019-12-30 | 2020-04-17 | 苏州珀罗汀生物技术有限公司 | Cell-free expression rhUTI protein system and method for producing rhUTI protein |
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