CN107236020A - A kind of method that simultaneously two albumen are carried out with specific marker or modification in same system - Google Patents
A kind of method that simultaneously two albumen are carried out with specific marker or modification in same system Download PDFInfo
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- CN107236020A CN107236020A CN201710447752.6A CN201710447752A CN107236020A CN 107236020 A CN107236020 A CN 107236020A CN 201710447752 A CN201710447752 A CN 201710447752A CN 107236020 A CN107236020 A CN 107236020A
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- Prior art keywords
- intein
- modification
- types
- specific marker
- leu
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
- C07K1/113—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/13—Labelling of peptides
Abstract
The present invention relates to a kind of method that simultaneously two albumen are carried out with specific marker or modification in same system, there is the advantage of extremely short N intein or C intein small fragments using two new intein of S1 types and S11 types, pass through the artificial synthesized intein small fragments, and plan modification group is introduced during synthesis, plan modification group is marked on to the N-terminal or C-terminal of target protein subsequently through trans-splicing.The present invention is simple to operate, and labeling effciency is high, has important application value in protein labeling field.
Description
Technical field
The invention belongs to the mark of protein labeling with modifying in field, more particularly to a kind of same system simultaneously to two
The method that albumen carries out specific marker or modification.
Background technology
The mark of protein labeling and modification, have important position in protein research application field.Such as to protein
Carry out visual marker and can be used for protein spike, the dynamic change of observing protein, to be visited to protein structure function
Rope；The stability that functional modification can be used for Protein requirement medicine is carried out to pharmaceutical grade protein class, extends its half-life period etc..Pass
The protein labeling or modification means of system utilize thing to be marked and Amino Acids in Proteins residue side mainly by chemical reaction
Chain radical reaction, so that thing to be marked is made an addition on target protein.But the above method is often without specificity, because egg
There may be multiple identical amino acid residues in white matter, cause to mark multiple sites, the mark effect of influence target protein
Really.
In order to solve this problem, the protein labeling technology of protein intron mediation obtains in-depth study with answering
With the specific marker of target protein can be realized by trans-splicing technology.Especially novel protein introne S1 with
The appearance of S11 types, makes the specific marker technology of protein obtain further deeply.S1 types intein N-intein fragments are only
Containing 11 amino acid, S11 types intein C-intein fragments are only containing 6 amino acid, and so short intein fragments can
To be synthesized by artificial chemistry, and thing to be marked can be introduced during synthesis, pass through follow-up protein trans-splicing
By substance markers to be marked on target protein.
Studies have reported that, label locus specificity can be marked on by target protein by novel protein introne
N-terminal or C-terminal, but be only often to realize simple target albumen in vitro for the research such as structure function of target protein
Mark, with certain limitation.Protein plays certain function, such as signal often by being acted synergistically between each albumen
Protein-interacting, antigen antibody interaction in path etc., need while research two is even multiple in this case
Target protein, to explore whole path or system.Therefore, multiple target proteins are marked in same system simultaneously for exploring albumen
The research such as interaction between matter is particularly important.If two or more new intein, it be able to can be sent out in same system
Raw montage reaction, and the montage effect that is independent of each other, then can realize multiple target proteins by different intein trans-splicing
Specific position is marked.This realizes that the specific position mark of multiple target proteins provides a kind of effective new side for same system
Method.
The content of the invention
The technical problems to be solved by the invention are to provide in a kind of same system carries out specificity to two albumen simultaneously
Mark or the method for modification, this method are simple to operate, and labeling effciency is high, has important application valency in protein labeling field
Value.
The method that simultaneously two albumen are carried out with specific marker or modification in a kind of same system of the present invention, including：
(1) target protein 1 is connected construction of fusion protein 1 with S1 types intein C-intein fragments；By target protein 2
Be connected construction of fusion protein 2 with S11 types intein N-intein fragments；
(2) chemical synthesis S1 types intein N-intein fragments add linker sequence GGG in its N-terminal simultaneously,
Linker first amino acid G introduces specific marker 1 or modification 1, obtains synthesizing fragment 1；Chemical synthesis S11 types intein's
C-intein fragments add linker sequence C FNK in its C-terminal simultaneously, and specific marker is introduced in linker most end amino acid K
2 or modification 2, obtain synthesize fragment 2；
(3) fusion protein 1, synthesis fragment 1, fusion protein 2 and synthesis fragment 2 are mixed in same system；Pass through S1
Type intein trans-splicing, the labeled upper specific marker 1 of N-terminal of fusion protein 1 or modification 1, pass through S11 types intein's
Trans-splicing, the labeled upper specific marker 2 of C-terminal of fusion protein 2 or modification 2.
Target protein 1 and target protein 2 in the step (1) are protease, polypeptide, protein drug or antibody.
Fusion expression plasmid can be built by gene cloning, target protein is connected with intein large fragment and carries out merging table
Reach.
The N-intein fragments of the S1 types intein include 11 amino acid, easily carry out artificial chemistry synthesis；S1 types
Intein C-intein fragments are used for the purifying of fusion protein 1 inside it containing 6His-tag；S11 types intein C-
Intein fragments include 6 amino acid, easily carry out artificial chemistry synthesis；S11 types intein N-intein fragments contain 6His-
Tag is used for the purifying of fusion protein 2.
S1 type intein and S11 types intein can independently carry out montage reaction in same system, no cross reaction, mutually
Montage effect is not influenceed；And generation montage in complicated system (such as cell pyrolysis liquid) can be mixed in height.
The S1 types intein is RBS1intein, particular sequence such as SEQ ID NO.1 (N-intein) and SEQ ID
Shown in NO.2 (C-intein)；Or SGS1intein, particular sequence such as SEQ ID NO.5 (N-intein) and SEQ ID
Shown in NO.6 (C-intein).
The S11 types intein is TE3S11intein, particular sequence such as SEQ ID NO.3 (N-intein) and SEQ ID
Shown in NO.4 (C-intein)；Or SGS11intein, particular sequence such as SEQ ID NO.7 (N-intein) and SEQ ID
Shown in NO.8 (C-intein).
Specific marker 1 and specific marker 2 in the step (2) are polypeptide or fluorescence labeling；Modification 1 and modification 2
For phosphorylation modification or acetylation modification.
The linker sequences GGG only has 3 amino acid, and length is extremely short, and the free amino that first G is present, and is easy to
It is connected with chemical modification；Linker sequence Cs last amino acid of FNK K has free amino, can be connected with chemical modification；
Linker sequences thereon can introduce different according to the increase that length is carried out the need for mark during chemical synthesis
Chemical modification, is marked on target protein by modification subsequently through intein trans-splicing.
Beneficial effect
The present invention can realize the specific marker of two target proteins of same system, and RBS1 and TE3S11intein
Montage efficiency has preferable labeling effciency up to more than 50%；And two intein small fragment it is very short, easily enter pedestrian
Work chemical synthesis, therefore a variety of marks or trim can be introduced during synthesis, provided for the mark of target protein
It is more alternative；Two target proteins can carry out different specific positions through the inventive method and mark, and be protein
Repercussion study, structure function research etc. provide feasible method or approach.
Brief description of the drawings
Figure 1A is precursor protein and montage product schematic diagram in montage reaction system；
Figure 1B is the SDS-PAGE and fluorescent scanning testing result of montage reaction system；
Fig. 1 C are SDS-PAGE the and Western-blot testing results of montage reaction system.
Embodiment
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention.In addition, it is to be understood that after the content of the invention lectured has been read, people in the art
Member can make various changes or modifications to the present invention, and these equivalent form of values equally fall within the application appended claims and limited
Scope.
Embodiment 1
Respectively to Trx and MBP target proteins (particular sequence such as SEQ ID NO.9 (MBP) and SEQ ID in same system
Shown in NO.10 (Trx)) carry out specific FITC and Rhodamine B fluorescence labelings：
Using the pet-32a after modification as carrier, Insert Fragment RBS1 C-intein and Trx build fusion expression plasmid
PMRBS1C, for expressed fusion protein RBS1C-Trx；Simultaneously using the pMAL after modification as carrier, Insert Fragment MBP and
TE3S11 N-intein, builds fusion expression plasmid pMTE3S11N, for expressed fusion protein MBP-TE3S11N；
By recombinant plasmid transformed host e. coli BL21, the flat board of amicillin resistance is incubated at, 37 DEG C were cultivated
Night；Picking monoclonal is inoculated in 3ml LB culture mediums (ampicillin of the 100ug/ml containing final concentration) afterwards, 37 DEG C, 200rpm
Overnight incubation；Afterwards with 1:100 ratios are transferred in 50ml LB culture mediums (ampicillin of the 100ug/ml containing final concentration), and 37
DEG C, 200rpm cultivated to OD600For 0.6~0.8 when, add final concentration 0.3mM IPTG, inducible protein expression, 20 DEG C,
200rpm overnight incubations；Thalline after 4000rpm collection incubated overnights, addition 10ml lysis buffer (20mM Tris,
300mM NaCl, pH8.0) thalline is resuspended；The thalline of resuspension is subjected to ultrasonication, 10000rpm, 4 DEG C supernatant is collected by centrifugation；
It is standby with reference to ni-sepharose purification method protein of interest；
Chemical synthesis RBS1intein N-intein small fragments add linker sequence GGG in its N-terminal simultaneously,
Linker first amino acid G introduces FITC marks, obtains carrying the RBS1N of FITC marks；Chemical synthesis TES311 C-
Intein small fragments add linker sequence C FNK in its C-terminal simultaneously, and Rhodamine is introduced in linker most end amino acid K
B is marked, and obtains carrying the TE3S11C of Rhodamine B marks；
The polypeptide of target protein after purification and the carrying fluorescence of synthesis (is carried into RBS1N and the carrying of FITC marks
The TE3S11C of Rhodamine B marks) mixing progress montage reaction, to mark target protein, included in montage reaction
2mM DTT；Then SDS-PAGE, Western-blot are carried out to montage reaction system and fluorescent scanning is detected；By RBS1 and
TE3S11intein specific montage reaction, the target protein Trx upper FITC fluorophors of N-terminal mark, and MBP C-terminal is then
Rhodamine B fluorophors on specific mark, as shown in Figure 1.From Figure 1B, target protein MBP C-terminal is special
Rhodamine B on the mark of property, Trx N-terminal is by FITC on specific mark.From Fig. 1 C, with fluorescent scanning result
It is corresponding, montage product MBP-Rhodamine B and FITC-Trx successfully be detected by specific antibody (anti-Trx and MBP).
SEQUENCE LISTING
<110>Donghua University
<120>A kind of method that simultaneously two albumen are carried out with specific marker or modification in same system
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<170> PatentIn version 3.3
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Claims (6)
1. simultaneously two albumen are carried out with the method for specific marker or modification in a kind of same system, including：
(1) target protein 1 is connected construction of fusion protein 1 with S1 types intein C-intein fragments；By target protein 2 with
The connected construction of fusion protein 2 of S11 types intein N-intein fragments；
(2) chemical synthesis S1 types intein N-intein fragments add linker sequence GGG in its N-terminal simultaneously, in linker
First amino acid G introduce specific marker 1 or modification 1, obtain synthesize fragment 1；Chemical synthesis S11 types intein C-
Intein fragments add linker sequence C FNK in its C-terminal simultaneously, and specific marker 2 is introduced in linker most end amino acid K
Or modification 2, obtain synthesizing fragment 2；
(3) fusion protein 1, synthesis fragment 1, fusion protein 2 and synthesis fragment 2 are mixed in same system；Pass through S1 types
Intein trans-splicing, the labeled upper specific marker 1 of N-terminal of fusion protein 1 or modification 1, pass through the anti-of S11 types intein
Formula montage, the labeled upper specific marker 2 of C-terminal of fusion protein 2 or modification 2.
2. simultaneously two albumen are carried out with the side of specific marker or modification in a kind of same system according to claim 1
Method, it is characterised in that：Target protein 1 and target protein 2 in the step (1) are protease, polypeptide, protein drug or
Antibody.
3. simultaneously two albumen are carried out with the side of specific marker or modification in a kind of same system according to claim 1
Method, it is characterised in that：The N-intein fragments of the S1 types intein include 11 amino acid；S1 types intein C-intein
Fragment is used for the purifying of fusion protein 1 inside it containing 6His-tag；S11 types intein C-intein fragments include 6
Amino acid；S11 types intein N-intein fragments, which contain 6His-tag, is used for the purifying of fusion protein 2.
4. specific marker or modification are carried out to two albumen simultaneously in a kind of same system according to claim 1 or 3
Method, it is characterised in that：The S1 types intein is RBS1 intein, particular sequence such as SEQ ID NO.1 and SEQ ID
Shown in NO.2；Or SGS1intein, particular sequence is as shown in SEQ ID NO.5 and SEQ ID NO.6.
5. specific marker or modification are carried out to two albumen simultaneously in a kind of same system according to claim 1 or 3
Method, it is characterised in that：The S11 types intein is TE3S11 intein, particular sequence such as SEQ ID NO.3 and SEQ
Shown in ID NO.4；Or SGS11 intein, particular sequence is as shown in SEQ ID NO.7 and SEQ ID NO.8.
6. simultaneously two albumen are carried out with the side of specific marker or modification in a kind of same system according to claim 1
Method, it is characterised in that：Specific marker 1 and specific marker 2 in the step (2) are polypeptide or fluorescence labeling；Modify 1 He
Modification 2 is phosphorylation modification or acetylation modification.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710447752.6A CN107236020A (en) | 2017-06-14 | 2017-06-14 | A kind of method that simultaneously two albumen are carried out with specific marker or modification in same system |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710447752.6A CN107236020A (en) | 2017-06-14 | 2017-06-14 | A kind of method that simultaneously two albumen are carried out with specific marker or modification in same system |
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| Publication Number | Publication Date |
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| CN107236020A true CN107236020A (en) | 2017-10-10 |
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ID=59987400
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| CN201710447752.6A Pending CN107236020A (en) | 2017-06-14 | 2017-06-14 | A kind of method that simultaneously two albumen are carried out with specific marker or modification in same system |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008083493A1 (en) * | 2007-01-10 | 2008-07-17 | University Of Saskatchewan | Stabilization of cyclic peptide structures |
| WO2009132455A1 (en) * | 2008-04-30 | 2009-11-05 | Paul Xiang-Qin Liu | Protein splicing using short terminal split inteins |
| CN104568861A (en) * | 2013-10-25 | 2015-04-29 | 华东理工大学 | Method for detecting protein interaction based on bimolecularly complemented covalent modification mark |
| CN105925596A (en) * | 2016-02-23 | 2016-09-07 | 上海交通大学 | Synthesis method of intein-based medicinal recombinant protein |
-
2017
- 2017-06-14 CN CN201710447752.6A patent/CN107236020A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008083493A1 (en) * | 2007-01-10 | 2008-07-17 | University Of Saskatchewan | Stabilization of cyclic peptide structures |
| WO2009132455A1 (en) * | 2008-04-30 | 2009-11-05 | Paul Xiang-Qin Liu | Protein splicing using short terminal split inteins |
| CN104568861A (en) * | 2013-10-25 | 2015-04-29 | 华东理工大学 | Method for detecting protein interaction based on bimolecularly complemented covalent modification mark |
| CN105925596A (en) * | 2016-02-23 | 2016-09-07 | 上海交通大学 | Synthesis method of intein-based medicinal recombinant protein |
Non-Patent Citations (3)
| Title |
|---|
| YING LIN，ET AL.: "Protein Trans-Splicing of Multiple Atypical Split Inteins Engineered from Natural Inteins", 《PLOS ONE》 * |
| 宋慧玲: "新型断裂蛋白质内含子介导的蛋白质反式剪接系统的建立及应用", 《中国优秀博士学位论文全文数据库 基础科学辑》 * |
| 张晓玲: "断裂蛋白质内含子介导的蛋白质两端标记", 《中国优秀硕士学位论文全文数据库 基础科学辑》 * |
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