CN109957559A - A kind of ubiquitin-specific protease 7 and its preparation method and application of fixed point label - Google Patents
A kind of ubiquitin-specific protease 7 and its preparation method and application of fixed point label Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
Abstract
The invention discloses a kind of ubiquitin-specific proteases 7 and its preparation method and application of fixed point label, after the ubiquitin-specific protease 7 carries out rite-directed mutagenesis, fixed point label is carried out using the unnatural amino acid with labelling groups, wherein, the unnatural amino acid with labelling groups is by tRNA synzyme/tRNA to introducing；The present invention passes through optimization protein expression process, select specific label and expression vector, and by tRNA synzyme and tRNA to introducing unnatural amino acid, each each condition of step is synergistic, it improves the expression quantity of albumen and completes fixed point label, it is easy to operate, it is easy to spread, the structure and activity for not influencing protease 7 have broad application prospects and huge market value.
Description
Technical field
The present invention relates to field of biotechnology more particularly to a kind of ubiquitin-specific proteases 7 and its system of fixed point label
Preparation Method and application.
Background technique
Ubiquitin is widely present in eucaryote, is played the part of in cell physiological biochemical process and is played an important role.In ubiquitin at different levels
Under the catalytic action of enzyme, the posttranslational modification that ubiquitin molecule can carry out specificity to target protein is called ubiquitination.Ubiquitination is
One important channel of protein post-translational modification cascades enzyme spcificity by 2 to 4 and modifies the stability of target protein, positioning
And activity.In contrast, ubiquitination process can be reversed by deubiquitinating enzymes, referred to as deubiquitination, i.e., single poly- and more by excision
Poly- ubiquitin acts on to fight ubiquitination, to provide the post-translational control of an extra level.Deubiquitinating enzymes can be specifically
Ester bond, peptide bond or the isopeptide bond formed between hydrolysis Ubiquitin C-terminal and target protein makes ubiquitin be detached from target protein, to participate in adjusting
Control cell cycle progression, protein degradation, gene expression, DNA reparation and Apoptosis etc. play an important role.
Ubiquitin-specific protease (ubiquitin-specific proteases, USPs) be currently known remove ubiquitin
Change enzyme family in member at most, the most multifarious one kind of structure, wide participation to cell cycle, oncogene and tumor suppressor gene etc.
In adjusting in relation to albumen.Abnormal deubiquitination activity is related with many diseases, including cancer.Ubiquitin-specific protease 7
(USP7) it is also referred to as protease (the Herpesvirusassociated of the adjoint ubiquitin-specific of Herpesvirus
Ubiquitin specific protease, HAUSP), it is one of important member of this family.Mankind USP7 is by 1102
Amino acid composition, and mouse USP7 is made of 1103 amino acid, molecular weight is about 135kDa.USP7 includes four structural domains, N-
The region amino terminal TRAF, catalysis region and 64kDa three regions such as the carboxy-terminal end region C- through being identified.N-
The region amino terminal TRAF is responsible for directly combining with substrate protein (such as p53, MDM2), main to influence between albumen and albumen
Interaction.Catalysis region includes two highly conserved histidine boxes and cysteine box, is ubiquitin protein enzyme family
Important feature.Ubiquitin binding pocket is located at catalytic domain, and the formation of ubiquitin binding pocket, can activate USP7, thus promote USP7 with
Ubiquitin combines, and plays the role of deubiquitination.The carboxy-terminal end region C- ubiquitin ligase containing there are two, can be with ubiquitin phase interaction
With and 5 continuous ubiquitin-like areas (Ubl).Originally cell protein 0 that it is used as a hsv protein to infect
(ICP0) binding companion is identified.Then, many potential substrates and binding molecule are identified.Wherein, ubiquitin
The substrate of some more features of specific protease 7 is in tumor suppression, DNA reparation, immune response, virus replication and apparent something lost
Transmission control etc. plays an important role.
The mode of protein labeling can be divided into non-fixed point label and fixed point label.Non- fixed point label refers to that probe does not have choosing
Selecting property and specifically combined with protein, probe acts on the multiple sites or region of protein, as Coomassie brilliant blue with
The combination of protein；Fixed point label refers to a certain position for being marked on protein that probe can be specific.Protein chemistry mark
The means of note are varied, mainly include amino acid tag, cysteine-marking sulfhydryl, introduce unnatural amino acid label, melt
Close the modes such as tag expression.Amino (- NH on protein2) be a kind of more active functional group, on amino nitrogen-atoms have compared with
Nucleophilic substitution reaction can occur for strong compatibility.Amino on protein is mostly derived from the N-terminal of lysine and protein, in alkali
In property solution, the amino reactive species of protein are various, fluorophor can by with NHS ester, isocyanate, isothiocyanic acid
Label is realized in the reaction of the groups such as vinegar, EDC, epoxide.The label of sulfydryl is active highest in native protein on protein
Functional group, have very strong compatibility, in the reaction of protein labeling, the reaction by sulfydryl (- SH) is most commonly seen
Mark mode, sulfydryl can react with groups such as disulfide, α-halogenatedacetamide, maleic amides.In probe pair
Before protein sulfhydryl is marked, in order to avoid the sulfydryl on protein itself forms the progress that disulfide bond hinders label reaction,
Albumen needs in the environment in reduction, it is often necessary to reagent such as dithiothreitol (DTT) (DTT), three (the 2- carboxylic second of reproducibility be added
Base) reagents such as phosphine (TCEP) are handled.These reagents are sloughed before label reaction carries out, in order to avoid these reducing agents and albumen
Sulfydryl competitive reaction in matter.Content relative to the lysine for possessing amino group, the cysteine containing sulfydryl is less, holds
Easily realize the label of single site.But at the lysine and cysteine all more than one often exposed due to protein and chemical ring
Border is often more similar, therefore the probe of amino, sulfydryl specific marker is often difficult to realize ideal selectivity.In larger level
In the research of (such as cell level), the problem of selectivity is less prominent, and to parse protein structure and dynamics is this kind of more
When the problem of fine level, the specific and better mark mode of selectivity is often just needed.
Meanwhile ubiquitin-specific protease 7 is marked in the presence of the problem that do not express or expression quantity is low to the fixed point of the albumen
It creates great difficulties, therefore it provides a kind of be able to solve the low problem of 7 expression quantity of ubiquitin-specific protease and complete its fixed point
The method of label has huge scientific research value and application prospect.
Summary of the invention
In view of the deficiencies of the prior art and actual demand, the present invention provide a kind of ubiquitin-specific albumen of fixed point label
Enzyme 7 and its preparation method and application, the present invention select specific label and expression vector by optimization protein expression process, and
By tRNA synzyme and tRNA to unnatural amino acid is introduced, each each condition of step is synergistic, improves the expression of albumen
Fixed point label is measured and completed, is had broad application prospects and huge market value.
To achieve this purpose, the present invention adopts the following technical scheme:
On the one hand, the present invention provides a kind of ubiquitin-specific protease 7 of fixed point label, the ubiquitin-specific protease
After 7 carry out rite-directed mutagenesis, fixed point label is carried out using the unnatural amino acid with labelling groups, wherein the ubiquitin specific
Property protease 7 have Strep label, the unnatural amino acid with labelling groups passes through tRNA synzyme/tRNA to drawing
Enter.
In the present invention, the unnatural amino acid can be by tRNA synzyme/tRNA to identification, and tRNA synzyme/tRNA
To codon corresponding with rite-directed mutagenesis is had, can be combined with the protease 7 after rite-directed mutagenesis.
Preferably, the ubiquitin-specific protease 7 passes through BL21 (DE3) CodonPlus and/or Rosetta gami bacterium
Strain is expressed, preferably BL21 (DE3) CodonPlus bacterial strain.
For inventor during studying protein fixed point label, that there are expression quantity is low for discovery ubiquitin-specific protease 7
The problem of not expressing even, the fixed point for completing the albumen, which marks, to be caused greatly to perplex, and inventor is by sufficiently investigating albumen
The various methods of matter fixed point label, summarize advantage and disadvantage, select through tRNA synzyme/tRNA to the side for introducing unnatural amino acid
Method realizes fixed point label, while optimizing the experiment flow of protein expression and label, screens a variety of expression vectors and label, final to send out
Now only after introducing unnatural amino acid and in conjunction with specific expression vector and label, each each condition of step is synergistic,
It is able to achieve the great expression of albumen and completes fixed point label.
Preferably, the labelling groups are selected from but not limited to alkynyl and/or azido.
The labelling groups are only modified in unnatural amino acid, have no influence to technical solution, in principle all marks
Note group is ok.
Preferably, the unnatural amino acid includes to azidophenylalanine and/or azido alanine.
Preferably, it is UAG that the rite-directed mutagenesis, which is by three company's codon mutations of the non-active region of ubiquitin protein enzyme 7,.
In the present invention, the UAG is the terminator codon being of little use, and is synthesized for the tRNA of codon corresponding with carrying
Enzyme/tRNA is combined.
Preferably, the ubiquitin-specific protease 7 further includes histidine tag, for use in purifying.
Second aspect, the present invention provide a kind of ubiquitin-specific protease 7 prepared as described in relation to the first aspect, including as follows
Step:
(1) non-active region of ubiquitin-specific protease 7 is subjected to rite-directed mutagenesis, while in ubiquitin-specific protease
7 both ends introduce histidine tag and Strep label respectively；
(2) ubiquitin-specific protease 7 that step (1) obtains is inserted on prokaryotic expression carrier；
(3) plasmid of the tRNA synzyme for the carrier mutation corresponding with carrying that step (2) obtains/tRNA pairs is converted to table
Up in strain, wherein the carrier and plasmid have different resistances, to screen；
(4) bacterial strain after screening is added in culture medium and carries out inducing expression, and be added can be transferred into tRNA synzyme/
TRNA is to identification and with the unnatural amino acid for marking required group.
In the present invention, the resistance that the plasmid of corotation needs to have different, so that a variety of antibiosis are added simultaneously in the medium
Element is screened, it is ensured that contains several plasmids being all transferred in the bacterial strain (or cell) screened.Screening obtains corotation
After bacterial strain (or cell), it is added in culture medium and carries out inducing expression.During expression need that corresponding unnatural amino acid is added, it should
TRNA synzyme/tRNA that unnatural amino acid can be transferred into is transported to peptide chain in the process to identification, protein translation, and
And the unnatural amino acid carry succeeding marker needed for group, such as alkynyl, azido etc., by protein purification come out after, obtain
To 7 albumen of ubiquitin-specific protease then have a group in the position of design mutation, can be by the group to obtaining
Ubiquitin-specific protease 7 carry out fixed point label.
In the present invention, the non-active region is the region other than the enzyme activity site of protease 7.
Preferably, the C-terminal of step (1) ubiquitin-specific protease 7 introduces histidine tag for purifying, and N-terminal draws
Enter Strep label.
Preferably, it is that connect codon mutations for the three of non-active region be UAG that step (1), which states rite-directed mutagenesis,.
Preferably, step (2) prokaryotic expression carrier is pET21a.
Preferably, step (3) the expression strain is BL21 (DE3) CodonPlus.
Preferably, step (4) labelling groups are selected from but not limited to alkynyl and/or azido.
Preferably, step (4) described unnatural amino acid includes to azidophenylalanine and/or azido alanine；
Described shown in formula I to azidophenylalanine, the azido alanine is as shown in Formula II:
As optimization technique method, a kind of ubiquitin-specific protease 7 prepared as described in relation to the first aspect, specifically include as
Lower step:
(1) rite-directed mutagenesis is carried out in the non-active region of ubiquitin-specific protease 7, by three Lian Mi of non-active region
Numeral sports UAG, while introducing histidine tag for purifying in C-terminal, and N-terminal introduces Strep label；
(2) ubiquitin-specific protease 7 for obtaining step (1) is inserted into prokaryotic expression carrier pET21a；
(3) plasmid of the tRNA synzyme for the carrier mutation corresponding with carrying that step (2) obtains/tRNA pairs is converted to table
Up in strain BL21 (DE3) CodonPlus, wherein the carrier and plasmid have different resistances, to screen；
(4) bacterial strain after screening is added in culture medium and carries out inducing expression, and be added can be transferred into tRNA synzyme/
TRNA is to identification and with the unnatural amino acid for marking required group.
The third aspect, the ubiquitin-specific protease 7 that the present invention provides a kind of fixed point label as described in relation to the first aspect are used
In preparing external diagnosis reagent case and/or drug.
Compared with prior art, the invention has the following beneficial effects:
(1) preparation method provided by the invention is easy to operate compared with chemical coupling method, does not need to handle using reducing agent
Albumen, the position of label and quantity are controllable, by selecting specific expression vector and label, in conjunction with the non-natural ammonia of introducing
Base acid, finally improves protein yield, small to the structure and activity influence of ubiquitin-specific protease 7；
(2) ubiquitin-specific protease 7 of fixed point label provided by the invention can be used in, and have wide application prospect
And market value.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of ubiquitin-specific protease 7 of the invention；
Fig. 2 is that unnatural amino acid of the invention expresses experimental principle figure；
Fig. 3 is 7 fixed point marking method of the ubiquitin-specific protease signal of the invention based on unnatural amino acid expression
Figure；
Fig. 4 is protein expression result figure of the invention.
Specific embodiment
Further to illustrate technological means and its effect adopted by the present invention, below in conjunction with attached drawing and by specific real
Mode to further illustrate the technical scheme of the present invention is applied, but the present invention is not limited in scope of embodiments.
Embodiment 1
(1) rite-directed mutagenesis is carried out to protease 7 by design primer, and introduces histidine tag in C-terminal and is used to purify, N
End introduces Strep label, introduces NdeI and XhoI restriction enzyme site respectively at the end 5' and the end 3', is obtained by PCR with mutation position
The segment of point, then overlap PCR is carried out, obtain complete 7 mutant nucleotide sequence of ubiquitin-specific protease, ubiquitin-specific albumen
The structural schematic diagram of enzyme 7 is as shown in Figure 1；
A) PCR reaction system is as follows:
The nucleic acid sequence of forward primer F1 is as shown in SEQ ID NO:1；
SEQ ID NO:1AGTCCATATGAACCACCAGCAGCAGCAG；The nucleic acid sequence of reverse primer R1 such as SEQ ID
Shown in NO:3:
SEQ ID NO:3AGTCCTCGAGGTTATGGATTTTAATGGCC；Above-mentioned PCR system obtains WT USP7-his piece
Section.
B) PCR reaction system is as follows:
The nucleic acid sequence of forward primer F2 is as shown in SEQ ID NO:2；
SEQ ID NO:2
AGTCCATATGTGGTCTCACCCACAGTTCGAAAAAGGCAGCAACCACCAGCAGCAGCAG；
The nucleic acid sequence of reverse primer R1 is as shown in SEQ ID NO:3:
SEQ ID NO:3AGTCCTCGAGGTTATGGATTTTAATGGCC；
Above-mentioned PCR system obtains strep tag-WT USP7-his segment.
C) PCR reaction system is as follows
The nucleic acid sequence of forward primer F2 is as shown in SEQ ID NO:2；
SEQ ID NO:2
AGTCCATATGTGGTCTCACCCACAGTTCGAAAAAGGCAGCAACCACCAGCAGCAGCAG；
The nucleic acid sequence of reverse primer overlap-R-K281UAA is as shown in SEQ ID NO:4:
SEQ ID NO:4CCCAAATGACTATGTTAACTTTTTTGTTCCT；
Above-mentioned PCR system obtains strep tag-USP7mutant segment.
D) PCR reaction system is as follows:
The nucleic acid sequence of forward primer overlap-F-K281UAA is as shown in SEQ ID NO:5；
SEQ ID NO:5AAGTTAACATAGTCATTTGGGTGGGAAACT；
The nucleic acid sequence of reverse primer R1 is as shown in SEQ ID NO:3:
SEQ ID NO:3AGTCCTCGAGGTTATGGATTTTAATGGCC；
Above-mentioned PCR system obtains USP7mutant-his segment.
E) PCR reaction system is as follows:
The nucleic acid sequence of forward primer F2 is as shown in SEQ ID NO:2；
SEQ ID NO:2
AGTCCATATGTGGTCTCACCCACAGTTCGAAAAAGGCAGCAACCACCAGCAGCAGCAG；
The nucleic acid sequence of reverse primer R1 is as shown in SEQ ID NO:3:
SEQ ID NO:3AGTCCTCGAGGTTATGGATTTTAATGGCC；
Above-mentioned PCR system obtains strep tag-USP7mutant-his segment.
(2) end DNA fragmentation 5' and 3' that PCR is obtained is respectively provided with Nde I and Xho I restriction enzyme site, carrier pET21a
Also it is linearized with the digestion 1 hour of 37 DEG C of the two restriction enzymes, then uses T4 ligase by carrier and segment 16
DEG C connection overnight；
Connection product is transferred in DH5 α bacterial strain by 42 DEG C of heat shock 45s, coated plate, and 37 DEG C are incubated overnight.Second day from plate
Upper picking single colonie carries out bacterium colony PCR, obtains the colony inoculations of positive findings to the LB Liquid Culture containing ammonia benzyl chloramphenicol resistance
In base, 37 DEG C, 200rpm shakes bacterium and stays overnight, and then collects thallus upgrading grain, send sequencing, and sequencing is correct, obtains required plasmid；
(3) successful 7 plasmid of ubiquitin-specific protease will be sequenced and be transformed into competent cell by 42 DEG C of heat treatment 45s
In BL21 (DE3) CodonPlus, recovers and is applied on LB plate (containing ampicillin) through 37 DEG C of cultures and screened,
37 DEG C are incubated overnight.
Second day picking single colonie is seeded in 4mL fresh LB (containing ampicillin), in 37 DEG C, 200rpm
Overnight incubation is added in 400mL culture medium by 1:100 in 37 DEG C for second day, and 200rpm is expanded culture, and monitors its OD600
When for 0.4-0.6, IPTG (isopropylthiogalactoside, the Isopropyl β-D- of 0.5mg/mL is added
Thiogalactoside), in 16 DEG C of inducing expressions, and be added can be transferred into tRNA synzyme/tRNA to identification and with label
The unnatural amino acid of group needed for alkynyl is to azidophenylalanine, after expression overnight, is centrifuged, receives in 5000rpm, 15min
Collect thallus, unnatural amino acid expresses experimental principle figure as shown in Fig. 2, the ubiquitin-specific egg based on unnatural amino acid expression
White 7 fixed point marking method schematic diagram of enzyme is as shown in Figure 3.
(4) thallus is resuspended with PBS, the ultrasound for the use of power being then 222w releases bacterial cell disruption endobacillary
Soluble protein, until solution is clarified, 11000rpm, is centrifuged 15min by 4 DEG C, and supernatant precipitating is in charge of.It is heavy to be resuspended with 1mL PBS
It forms sediment, then 11000rpm, 4 DEG C, is centrifuged 15min, collects supernatant.
Embodiment 2
Compared with Example 1, other than the labelling groups of selection are azido, other conditions are same as Example 1.
Embodiment 3
Compared with Example 1, other than the unnatural amino acid of selection is azido alanine, other conditions and embodiment
1 is identical.
Embodiment 4
Compared with Example 1, other than selecting and expressing bacterial strain as Rosetta gami bacterial strain, other conditions and embodiment 1
It is identical.
Comparative example 1
Compared with Example 1, other than not introducing unnatural amino acid, other conditions are same as Example 1.
Comparative example 2
Compared with Example 1, in addition to not carrying Strep label, other conditions are same as Example 1.
Protein Detection
The embodiment 1-4 and comparative example 1-2 supernatant collected is in charge of carry out electrophoresis by SGS-PAGE, and is carried out
Western blot detects albumen.
Primary antibody uses source of mouse his antibody, and 1:10000,4 DEG C overnight.Then it is cleaned 3 times with 1 × TBST, each 5min；Two
It is anti-to use sheep anti-mouse antibody, 1:1000, room temperature, 1h.It is cleaned 3 times after recycling secondary antibody with 1 × TBST, then each 5min is used
ECL chemical imaging method test strip, as a result as shown in Figure 4；
As shown in Figure 4, comparative example 1-2 cannot achieve the expression of ubiquitin-specific protease 7, and embodiment 4 uses
The expression of Rosetta gami bacterial strain is weaker, is only introducing unnatural amino acid, carries Strep label and is selecting BL21
(DE3) CodonPlus expresses the common synergy of each condition of each step of bacterial strain, could promote mutually to realize the efficient of ubiquitin protein enzyme 7
Expression and fixed point label, the molecular size range of ubiquitin-specific protease 7 is about 135kD.
In conclusion the present invention is by selecting specific expression bacterial strain, carrying strep label and introducing non-natural amino
Acid, optimization experiment flow, each each condition of step is synergistic, finally realizes the high efficient expression of ubiquitin-specific protease 7 and determines
Point label, preparation method is succinctly easy to operate, influences on the activity of albumen and structure smaller, has broad application prospects and market
Value.
The Applicant declares that the present invention is explained by the above embodiments method detailed of the invention, but the present invention not office
Be limited to above-mentioned method detailed, that is, do not mean that the invention must rely on the above detailed methods to implement.Technical field
Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to each raw material of product of the present invention
Addition, selection of concrete mode etc., all of which fall within the scope of protection and disclosure of the present invention.
Sequence table
<110>Shenzhen Xianjin Technology Academe
<120>a kind of ubiquitin-specific protease 7 and its preparation method and application of fixed point label
<130> 2017
<141> 2017-12-26
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cccaaatgac tatgttaact tttttgttcc t 31
<210> 5
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Claims (10)
1. a kind of ubiquitin-specific protease 7 of fixed point label, which is characterized in that the ubiquitin-specific protease 7 is determined
After point mutation, fixed point label is carried out using the unnatural amino acid with labelling groups；
Wherein, the ubiquitin-specific protease 7 has Strep label；
The unnatural amino acid with labelling groups is by tRNA synzyme/tRNA to introducing.
2. ubiquitin-specific protease 7 according to claim 1, which is characterized in that the ubiquitin-specific protease 7 is logical
It crosses Rosetta gami bacterial strain and/or BL21 (DE3) CodonPlus bacterial strain is expressed, preferably BL21 (DE3)
CodonPlus bacterial strain.
3. ubiquitin-specific protease 7 according to claim 1 or 2, which is characterized in that the labelling groups include alkynyl
And/or azido；
Preferably, the unnatural amino acid includes to azidophenylalanine and/or azido alanine.
4. ubiquitin-specific protease 7 according to any one of claim 1-3, which is characterized in that the rite-directed mutagenesis
It is UAG to connect codon mutation for the three of the non-active region of ubiquitin protein enzyme 7.
5. ubiquitin-specific protease 7 described in any one of -4 according to claim 1, which is characterized in that the ubiquitin specific
Property protease 7 further includes histidine tag.
6. a kind of prepare ubiquitin-specific protease 7 according to any one of claims 1 to 5, which is characterized in that including such as
Lower step:
(1) non-active region of ubiquitin-specific protease 7 is subjected to rite-directed mutagenesis, while in ubiquitin-specific protease 7
Both ends introduce histidine tag and Strep label respectively；
(2) ubiquitin-specific protease 7 that step (1) obtains is inserted on prokaryotic expression carrier；
(3) plasmid of the tRNA synzyme for the carrier mutation corresponding with carrying that step (2) obtains/tRNA pairs is converted to expression bacterium
In kind, wherein the carrier and plasmid have different resistances, to screen；
(4) bacterial strain after screening is added in culture medium and carries out inducing expression, and be added and can be transferred into tRNA synzyme/tRNA pairs
Identification and the unnatural amino acid for having the required group of label.
7. according to the method described in claim 6, it is characterized in that, the C-terminal of step (1) ubiquitin-specific protease 7 is drawn
Enter histidine tag, N-terminal introduces Strep label.
8. method according to claim 6 or 7, which is characterized in that step (2) prokaryotic expression carrier is pET21a；
Preferably, it is UAG that step (1) described rite-directed mutagenesis, which is by three company's codon mutations of non-active region,；
Preferably, step (3) the expression strain is BL21 (DE3) CodonPlus；
Preferably, step (4) described labelling groups include alkynyl and/or azido；
Preferably, step (4) described unnatural amino acid includes to azidophenylalanine and/or azido alanine.
9. the method according to any one of right 6-8, which is characterized in that specifically comprise the following steps:
(1) rite-directed mutagenesis is carried out in the non-active region of ubiquitin-specific protease 7, connects codon for the three of non-active region
UAG is sported, while introducing histidine tag in C-terminal, N-terminal introduces Strep label；
(2) ubiquitin-specific protease 7 for obtaining step (1) is inserted into prokaryotic expression carrier pET21a；
(3) plasmid of the tRNA synzyme for the carrier mutation corresponding with carrying that step (2) obtains/tRNA pairs is converted to expression bacterium
In kind BL21 (DE3) CodonPlus, wherein the carrier and plasmid have different resistances, to screen；
(4) bacterial strain after screening is added in culture medium and carries out inducing expression, and the tRNA synzyme/tRNA that can be transferred into is added
To identification and with the unnatural amino acid for marking required group.
10. a kind of ubiquitin-specific protease 7 of fixed point label according to any one of claims 1 to 5 is used to prepare in vitro
Diagnostic kit and/or drug.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114195878A (en) * | 2021-12-21 | 2022-03-18 | 中国人民解放军军事科学院军事医学研究院 | Ubiquitin mutant and preparation method and application thereof |
| CN114250243A (en) * | 2020-09-24 | 2022-03-29 | 深圳华大生命科学研究院 | System and method for detecting aminoacyl-tRNA synthetase and tRNA activity in extreme halophilic organisms |
| CN114450295A (en) * | 2019-08-28 | 2022-05-06 | 敦提大学 | Activity-based probes |
| CN114250243B (en) * | 2020-09-24 | 2024-05-03 | 深圳华大生命科学研究院 | System and method for detecting aminoacyl-tRNA synthetase and tRNA activity in extreme halophilic organisms |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003189883A (en) * | 2001-09-28 | 2003-07-08 | Dai Ichi Seiyaku Co Ltd | New ubiquitin-specific protease |
| WO2006131370A1 (en) * | 2005-06-10 | 2006-12-14 | Novartis Ag | Protein crystal |
| CN102219841A (en) * | 2011-05-24 | 2011-10-19 | 中国科学院遗传与发育生物学研究所 | Phytomass-related protein as well as coding gene and application thereof |
| WO2012175924A2 (en) * | 2011-06-24 | 2012-12-27 | Medical Research Council | Incorporation of substituted lysines into polypeptides |
| CN104099360A (en) * | 2013-04-12 | 2014-10-15 | 北京大学 | Preparation for protein or peptide labeled by unnatural amino acid |
-
2017
- 2017-12-26 CN CN201711436434.6A patent/CN109957559A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003189883A (en) * | 2001-09-28 | 2003-07-08 | Dai Ichi Seiyaku Co Ltd | New ubiquitin-specific protease |
| WO2006131370A1 (en) * | 2005-06-10 | 2006-12-14 | Novartis Ag | Protein crystal |
| CN102219841A (en) * | 2011-05-24 | 2011-10-19 | 中国科学院遗传与发育生物学研究所 | Phytomass-related protein as well as coding gene and application thereof |
| WO2012175924A2 (en) * | 2011-06-24 | 2012-12-27 | Medical Research Council | Incorporation of substituted lysines into polypeptides |
| CN104099360A (en) * | 2013-04-12 | 2014-10-15 | 北京大学 | Preparation for protein or peptide labeled by unnatural amino acid |
Non-Patent Citations (4)
| Title |
|---|
| GEORGE J KLARMANN 等: "Site- and subunit-specific incorporation of unnatural amino acids into HIV-1 reverse transcriptase", 《PROTEIN EXPRESSION AND PURIFICATION》 * |
| TATJANA SCHNEIDER 等: "Dissecting ubiquitin signaling with linkage-defined and protease resistant ubiquitin chains", 《ANGEWANDTE CHEMIE》 * |
| 张晨: "UAA编码氨基酸表达体系的构建", 《中国优秀硕士学位论文全文数据库 基础科学辑》 * |
| 王梦云 等: "马立克氏病毒UL36蛋白的表达及其去泛素化酶活性分析", 《畜牧与兽医》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114450295A (en) * | 2019-08-28 | 2022-05-06 | 敦提大学 | Activity-based probes |
| CN114250243A (en) * | 2020-09-24 | 2022-03-29 | 深圳华大生命科学研究院 | System and method for detecting aminoacyl-tRNA synthetase and tRNA activity in extreme halophilic organisms |
| CN114250243B (en) * | 2020-09-24 | 2024-05-03 | 深圳华大生命科学研究院 | System and method for detecting aminoacyl-tRNA synthetase and tRNA activity in extreme halophilic organisms |
| CN114195878A (en) * | 2021-12-21 | 2022-03-18 | 中国人民解放军军事科学院军事医学研究院 | Ubiquitin mutant and preparation method and application thereof |
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