CN105061581B - Can gene code holoprotein catenne preparation method - Google Patents
Can gene code holoprotein catenne preparation method Download PDFInfo
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Abstract
The invention discloses it is a kind of can gene code holoprotein catenne preparation method, make that mutually winding occurs between protein monomers molecule using albumen interlocking structure unit, and catenne is formed to making protein monomers molecule that cyclisation occur using the albumen qualitative response similar to spy label spy hunter's coupling reaction pair, structure comprising the albumen qualitative response pair encoding gene corresponding with the recombinant protein of albumen interlocking structure unit and is introduced into vector plasmid first, then it is expressed in cell, purifying expression albumen, obtains protein catenne.The present invention utilizes the form of gene code, and the protein with catenne structure is relatively efficiently obtained in cell body, can farthest reduce the difficulty of protein catenne preparation, obtain the reactive protein of function-stable.
Description
Technical field
The present invention relates to the preparation of boiomacromolecule, more particularly to the preparation side that protein catenne is expressed in vivo
Method.
Background technology
Catenne is the supermolecule being made of molecule all linked with one another, has special mechanical interlock, is led in supermolecule
It is received significant attention in domain.In oversubscription subdomains, there are mainly two types of the methods for being typically prepared catenne：One is mutual using molecule
It winds and the possibility of cyclization is synthesized, this often yield is low based on statistical mode, poor controllability；Another kind is profit
Keep molecule pre-assembled with noncovalent interaction, target product (Gil-Ramirez, G. can be obtained with higher yield；
Leigh,D.A.；Stephens,A.J.Angew.Chem.Int.Ed.2015,54,6110-6150.).
Compared to the catenne in supermolecule, the special nature of protein catenne more concentrates in the raising of stability.Albumen
The stability of matter structure and function is of great significance for its bioactivity.Specific reactive protein, such as some enzymes, often
Harshness is required to preservation condition, and environmentally sensitive property is high, thus limited its scope of application.By changing opening up for protein
Structure is flutterred, such as protein is made to be cyclized, or the strategy such as formation intramolecular disulfide bond can effectively improve the stability of protein.It grinds
Study carefully and point out, the catenne of protein improves effective concentration, to significantly reduce the free energy correctly folded, stabilizes folding
Stack structure.The capsid protein of nature pnagus medius HK97 is that there are multiple catennes to form " hauberk " structure, has pole
High heat tolerance (Wikoff, W.R.；Liljas,L.；Duda,R.L.,et al.Science 2000,289,2129-
2133.).So if the catenne of specific protein can be realized, it is possible to realize and improve the various changes of substrate protein confrontation
Property condition tolerance, and then widen the substrate application range or reduce apply threshold.
The current report for preparing protein catenne is few, and one of example is to utilize tumor suppressor p53 structure lists
For the dimerization of member as intermolecular interlocking unit, the natural chemistry under vitro is connected to cyclic mode, is successfully prepared
Polypeptide catenne (Yan, L.Z.；Dawson,P.E.Angew.Chem.Int.Ed.2001,40,3625-3627.).However it is at present
Only, the report of the larger recombinant protein catenne of molecular weight is not prepared.Realize the catenne of recombinant protein, and thin
Protein catenne under born of the same parents' environment can farthest reduce the difficulty of preparation, while be possible to improve the adaptation of substrate
Property.
Invention content
In order to realize more efficient protein catenne, the present invention provides a kind of recombinant proteins in vivo
The method of (including in prokaryotic cell and in eukaryocyte) expression and catenne.
Realize that the catenne of in-vivo recombination protein, recombinant protein monomer molecule need to have two necessary spies
Sign：(1) it can mutually be wound between monomer molecule, form interlocking structure unit；(2) monomer molecule formed intramolecular covalent bonds from
And it is cyclized.For (1) a feature, it has been reported that some protein domains can be in cellular environment and vitro
It is lower to form the dimeric structure mutually wound, such as protein HP0242, DNA knot in tumor suppressor p53, helicobacter pylori
Hop protein Arc etc., the dimerization unit that can be wound as protein chain using these primitive structures.Meanwhile in recent years, protein
Reaction provides think of to a series of realization for developing into (2) a feature of posttranslational modification enzymes and specific protein qualitative response pair
Road, this includes transpeptidase, glutamine transaminage, spy label-spy hunter coupling reaction equity.Can be mentioned that spy
Label-spy hunter is derived from the albumen qualitative response pair of micrococcus scarlatinae, has fabulous biocompatibility, and be resistant to severe
The reaction environment at quarter, and extremely efficient (Zakeri, B.；Fierer,J.O.；Celik,E.,et
al.Proc.Natl.Acad.Sci.USA2012,109,E690-E697.).Spy label is one section of short peptide chain, and spy hunter is one
The structural domain of 13kD molecular weight, occurs Supramolecular Recognition and combination first between spy label and spy hunter, on spy label
Aspartic acid occurs coupling reaction under the action of self-catalysis with the lysine on spy hunter and forms amido bond in turn, to realize
Mutual is stably connected with.The reaction is to having been achieved with the cyclisation of elastin laminin analog and specific function protein
(Zhang,W.B.；Sun,F.；Tirrell,D.A.,et al.J.Am.Chem.Soc.2013,135,13988-13997；
Schoene,C.；Fierer,J.O.；Bennett,S.P.,et al.Angew.Chem.Int.Ed.2014,53,6101-
6104.), research of the invention, which finds them also, can be applied to prepare catenne.
Specifically, the technical scheme is that：
A kind of preparation method of protein catenne, includes the following steps：
1) protein catenne sequence monomer is designed, which includes at least the albumen being made of two peptide chains
Qualitative response pair and the albumen interlocking structure unit between two peptide chains, wherein the albumen interlocking structure unit makes albumen
Mutual winding between molecule occurs for matter catenne monomer, and the albumen qualitative response is formed to making protein catenne monomer that cyclisation occur
Catenne；
2) it builds the corresponding encoding gene of protein catenne monomer and is introduced into vector plasmid；
3) gene of step 2) structure is expressed in cell；
4) expression albumen is purified, obtains high-purity protein catenne.
Above-mentioned steps 1) described in albumen qualitative response to be preferably the reaction pair of spy label-spy hunter or its to maintain coupling anti-
The mutant of answering property, it can also be albumen qualitative responses pair that similar other are coupled principle based on recombination self-catalysis.
SEQ ID No in the amino acid sequence of typical spy label such as sequence table：Shown in 1, corresponding gene order can
To be SEQ ID No in sequence table：2, the degeneracy based on codon can also be the base of other same amino acid sequences of coding
Because of sequence；SEQ ID No in the amino acid sequence of corresponding spy hunter such as sequence table：Shown in 3, corresponding gene order can be with
It is SEQ ID No in sequence table：4 or the same amino acid sequence of coding other gene orders.
The spy label and/or the mutant of spy hunter refer to passing through 1 in spy label and/or the amino acid sequence of spy hunter
A or more amino acid replace, miss or add and derivative peptide chain, the amino acid replaced, missed or added are residual
Base will not have an impact their coupling reaction function.Amino acid residue is replaced, missed or added, and to correlation
The detection of function can be realized by the ordinary skill in the art.
In addition to spy label-spy hunter's tool pair, the albumen qualitative response is to can also be transpeptidase, glutamine transaminage
Deng.Transpeptidase can be in conjunction with particular sequence leucine-proline-arbitrary amino acid-threonine-glycine, and cuts off and wherein revive
Peptide bond between propylhomoserin and glycine, then it is catalyzed more glycine sequences formation peptide bond of the threonine with another molecule, to realize
It is covalently attached to.Glutamine transaminage can within catalytic proteins or between acyl group transfer reaction, realize between protein
Covalent cross-linking is also once used to realize cyclisation (Touati, the J. of polypeptide；Angelini,A.；Hinner,M.J.,et
al.ChemBioChem2011,12,38-42.).These protein tools are cyclized after protein catenne monomer can be made to express, shape
At catenne ring.The unit of albumen interlocking structure described in step 1) is preferably the egg in tumor suppressor p53, helicobacter pylori
White matter HP0242, DNA binding protein Arc and other can cause chain winding arrangement protein oligomerization domain, can be with
The cellular construction wound as protein chain using the oligomerization of these structures.Wherein tumor suppressor p53 oligomerization domains
Amino acid sequence such as sequence table in SEQ ID No：Shown in 5, corresponding gene order can be SEQ ID in sequence table
No：6, the degeneracy based on codon can also be the gene order of other same amino acid sequences of coding.
Further, it in addition to albumen qualitative response pair and albumen interlocking structure unit, can also be wrapped in protein catenne monomer
One or more identical or different functional protein structural domain is included, these functional protein structural domains can be located in catenne ring
And/or outside catenne ring.The functional protein can be elastin laminin analog (ELP), red fluorescent protein (mCherry), β-
Lactamase (BLA), dihyrofolate reductase (DHFR) etc. and medicine, agricultural, industry, scientific research field other albumen.
Below by taking spy label-spy hunter reaction pair and tumor suppressor p53 oligomerization domains as an example, to protein rope
The concrete structure design of hydrocarbon monomer illustrates.The basic structure of protein catenne monomer is following (a) or one of (b)：(a)
It is followed successively by spy label, tumor suppressor p53 oligomerization domains, spy hunter from N-terminal to C-terminal；(b) it is followed successively by from N-terminal to C-terminal
Spy hunter, tumor suppressor p53 oligomerization domains, spy label.
In the structure of above-mentioned (a) and (b), between spy label and tumor suppressor p53 oligomerization domains, and/
Or, can be inserted between spy hunter and tumor suppressor p53 oligomerization domains it is arbitrary one or more it is identical or
Different functional protein structural domains forms functional protein and is located at the protein catenne in catenne ring.For example, in the base of (a) structure
On plinth, the protein catenne monomer of functional protein is inserted into catenne ring can be but not limited to following several situations：(i) from N-terminal
It is followed successively by spy label, functional protein structural domain, tumor suppressor p53 oligomerization domains and spy hunter to C-terminal；(ii) from N
End to C-terminal is followed successively by spy label, tumor suppressor p53 oligomerization domains, functional protein structural domain and spy hunter；(iii)
It is followed successively by spy label, the first functional protein structural domain, tumor suppressor p53 oligomerization domains, the second work(from N-terminal to C-terminal
Energy protein structure domain and spy hunter also may be used wherein the first functional protein structural domain and the second functional protein structural domain can be identical
With difference.The functional protein structural domain is also not necessarily limited to be a structural domain, can also be multiple structural domains.For (b) structure,
Such functional protein can be equally carried out to be inserted into.
In addition, functional protein may be located on outside catenne ring, at (a) or (b) on architecture basics, add in N-terminal and/or C-terminal
Add one or more arbitrary identical or different functional protein structural domain.Such protein catenne monomer structure include but
It is not limited to following several situations：(I) functional protein structural domain, spy label, tumor suppressor p53 widows are followed successively by from N-terminal to C-terminal
Multimerisation domain and spy hunter；(II) spy label, tumor suppressor p53 oligomerization domains, spy are followed successively by from N-terminal to C-terminal
Hunter and functional protein structural domain；(III) the first functional protein structural domain, spy label, tumor suppression are followed successively by from N-terminal to C-terminal
Factor p53 oligomerization domains, spy label and the second functional protein structural domain, wherein the first functional protein structural domain and the second work(
Energy protein structure domain may be the same or different.In above-mentioned (I) to (III) any one structure, spy label and spy hunter
Position can be interchanged, and between spy label and tumor suppressor p53 oligomerization domains, and/or, spy hunter with it is swollen
One or more arbitrary functional protein structural domain can also be inserted between tumor inhibiting factor p53 oligomerization domains.
That is can have in catenne ring and simultaneously functional protein structural domain outside catenne ring.
The structure design of above-mentioned protein catenne monomer is with spy label-spy hunter reaction pair and tumor suppressor p53
It is illustrated for oligomerization domain, but this does not imply that can only be using spy hunter and spy label as albumen qualitative response pair
With using tumor suppressor p53 oligomerization domains as albumen interlocking structure unit, those skilled in the art can select to appoint
What can gene code albumen qualitative response to realizing the cyclisation of monomer molecule, and selection arbitrarily can gene code albumen it is mutual
Lock construction unit realizes the mutual winding of monomer molecule.
Above-mentioned steps 2) by the corresponding gene of recombinant DNA technology structure protein catenne monomer, and be introduced into suitably
In vector plasmid.The applicable carrier such as pQE and pET series plasmids of prokaryotic expression system.It is convenient for subsequent purifying, it can be with
His labels are added in the N-terminal or C-terminal of protein catenne monomer.
Above-mentioned steps 3) it is typically to import the plasmid of structure in colibacillus engineering to express the protein catenne list
Body, expression temperature can be the arbitrary temps between 16 DEG C -37 DEG C.According to actual needs, can also using eukaryotic expression system come
Express the protein catenne monomer.
Above-mentioned steps 4) in, common purification process is to carry out nickel for being added to the protein catenne monomer of His labels
Affinity chromatography.
The present invention protein catenne preparation method it is a technical advantage that can utilize gene code form, in cell
The protein with catenne structure is relatively efficiently obtained in vivo, can farthest reduce the difficulty of protein catenne preparation
Degree, obtains the reactive protein of function-stable.
Description of the drawings
Fig. 1 shows the expressions of the AEXEB of the measurement of embodiment 3 at different temperatures.
Fig. 2 shows the gel filtration chromatography data of crude product after AEXEB ni-sepharose purifications.
Fig. 3 shows the flight time mass spectrum data of AEXEB protein catennes.
Fig. 4 shows the data of AEXEB protein catenne digestions.
Fig. 5 shows the expressions of the EAXEB of the measurement of embodiment 3 at different temperatures.
Fig. 6 shows the gel filtration chromatography data of crude product after EAXEB ni-sepharose purifications.
Fig. 7 shows the flight time mass spectrum data of EAXEB protein catennes.
Fig. 8 shows the data of EAXEB protein catenne digestions.
Fig. 9 shows the cleavage map of AXB and BXA.
Figure 10 shows the flight time mass spectrum data of AXB (a) and BXA (b).
Specific implementation mode
Below by embodiment, further the present invention is described in detail, the model of but do not limit the invention in any way
It encloses.
Prepare protein catenne the specific steps are, by recombination engineering technology structure contain His6Label, spy are caught
Hand (A), tumor suppressor p53 oligomerization domains (X), elastin laminin analog (E) (or BLA, DHFR and
Other albumen such as mCherry) and spy hunter (B) fusion protein sequence plasmid, then by molecular cloning method by matter
Grain is transformed into expression in escherichia coli fusion protein, then the subject fusion of purifying is obtained by the protein purifications method such as affinity chromatography
Albumen.The subject fusion proteins include：AEXEB、EAXEB、AXB、BXA、AX-mCherry-B、AX-BLA-B、AX-DHFR-
B。
Utilize size exclusion chromatography, tobacco etch virus protease (TEV) digestion, substance assistant laser desorpted ionized flight
The means such as time mass spectrum characterize the molecular weight of AEXEB, EAXEB, the properties such as topological structure.
Involved detection means is as follows：
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) carries out sample analysis.
Size exclusion chromatography (SEC) uses the fast protein liquid chromatography (FPLC) of GE Healthcare companies.Make
Chromatographic column is Superdex 200increase 10/300GL.Mobile phase is PBS (10mM disodium hydrogen phosphates, 2mM phosphoric acid
Potassium dihydrogen, 137mM sodium chloride, 2.7mM potassium chloride, pH=7.4).
Embodiment 1：Build AEXEB and EAXEB fusion proteins plasmid and in expression in escherichia coli
SEQ ID No in AEXEB sequences such as sequence table constructed by the present embodiment：Shown in 7, wherein the 14th~26 is A,
29th~108 and 151~230 be E, the 111st~148 is X, and the 233rd~359 is B, and the 237th~243 is TEV enzymes
Enzyme site.
SEQ ID No in EAXEB sequences such as sequence table constructed by the present embodiment：Shown in 8, wherein the 96th~108 is
A, the 14th~93 and 154~233 be E, the 114th~151 is X, and the 238th~364 is B, and the 242nd~248 is TEV enzymes
Enzyme site.
The gene order of above-mentioned fusion protein is imported in carrier pQE-80L, Escherichia coli are then converted.
The expression in e. coli bl21 (DE3).Before extensive expression, the monoclonal bacterium for first obtaining conversion is inoculated with
(contain 100 μ g/mL ampicillins) in 10mL 2 × YT culture mediums, shake culture is stayed overnight under the conditions of 37 DEG C, 250rpm.The
(in 3L shaking flasks, contain 100 μ g/mL ampicillins) in the fresh 2 × YT culture mediums of two days 1L that transfer and carries out large-scale culture simultaneously
Induced expression.It is as follows：37 DEG C, shake culture about 2h, OD under the conditions of 200rpm600About 0.5 to 0.8, isopropyl is added
Base-β-D- thiogalactosides (IPTG), final concentration of 1mM, then in 180rpm, 16 DEG C of cultures are for 24 hours or after 37 DEG C of culture 4h
Collect thalline.
Embodiment 2：Build AXB and BXA fusion proteins plasmid and in expression in escherichia coli
SEQ ID No in AXB sequences such as sequence table constructed by the present embodiment：Shown in 9, wherein the 16th~28 is A, the
33~70 are X, and the 77th~203 is B, and the 81st~87 is TEV restriction enzyme sites.
SEQ ID No in BXA sequences such as sequence table constructed by the present embodiment：Shown in 10, wherein the 17th~143 is B, the
21~27 are TEV restriction enzyme sites, and the 156th~193 is X, and the 199th~211 is A.
The above protein sequence is structure in pQE-80L expression vectors.The then expression in e. coli bl21 (DE3).
Before extensive expression, first the monoclonal bacterium that conversion obtains is seeded in 5mL 2 × YT culture mediums and (contains 100 μ g/mL ammonia
Parasiticin), at 37 DEG C, shake culture is stayed overnight under the conditions of 250rpm, is transferred within second day in the fresh 2 × YT culture mediums of 1L
(being contained in the triangular flask of 3L, contain 100 μ g/mL ampicillins), at 37 DEG C, is cultivated on a large scale under the conditions of 250rpm,
Wait for its OD600It grows between 0.6-1.0, isopropyl-β-D-thiogalactoside (IPTG) is added and induces, final concentration of 0.1mM,
Then cultivation temperature is set as 16 DEG C, 220rpm, and thalline is collected after cultivating 20h.
Embodiment 3：Nickel affinity chromatography column purification AEXEB and EAXEB catenne protein
1L E. coli broths are collected in centrifugal bottle, thalline, the culture of removal upper layer are collected with 5000g centrifugal forces
Liquid.It is denaturalized buffer solution A (20mM disodium hydrogen phosphates, 500mM chlorinations with the 40mL containing 2% polysorbas20 and 1mM phenylmethylsulfonyl fluorides
Sodium, 25mM imidazoles, 8M urea, pH=8.0) be resuspended thalline, then with Ultrasound Instrument under the conditions of 4 DEG C smudge cells, then by large intestine
Bacillus breakdown products centrifuge 20 minutes under 4 DEG C, 28000g centrifugal force.Obtained supernatant is balanced with buffer solution A is denatured
Nickel affine resin mixing, stir 1h at 4 DEG C.Mixture is placed in void column, carries out gravity stream elution, and eluent is that denaturation is slow
Rush solution B (20mM disodium hydrogen phosphates, 500mM sodium chloride, 8M urea, 250mM imidazoles, pH=8.0).Elute obtained albumen
Matter tests purity with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).It analyzes on sample and 5 × protein
Sample buffer solution mixes, and after 98 DEG C of heating 10min, is loaded into 10% sodium dodecyl sulfate-polypropylene acrylamide gel, vertical electricity
70min (electrophoresis liquids are run under swimming 80-140V voltages：25mM trihydroxies aminomethane, 250mM glycine, 0.1% dodecyl
Sodium sulphate).Gel observes pillar location after being handled with Coomassie brilliant blue G-250 dyeing.Fig. 1 and Fig. 5 is shown at different temperatures
AEXEB and EAXEB albumen nickel affinity chromatography purify situation, by Bacillus coli expression, nickel affinity chromatography column purification can
Obtain the protein catenne of higher degree.
Embodiment 4：Nickel affinity chromatography column purification AXB and BXA catenne protein
1L Escherichia coli culture mediums are collected into centrifugal bottle, thalline is collected with 6000rpm centrifugal forces, abandons supernatant
Liquid.Wet bacterium weight is weighed, denatured lysis buffer (100mM disodium hydrogen phosphates, 10mM trihydroxy methyl amino is added according to 5mL/g
Methane, 10mM imidazoles, 8M urea, pH=8.0) thalline is resuspended, it, will resuspension thalline Ultrasound Instrument 4 after -80 DEG C of freeze thawing 1-2 time
Smudge cells under the conditions of DEG C, then by breakdown products at 4 DEG C, 25000g is centrifuged 30 minutes.According to volume in obtained supernatant
1:Nickel affine resin is added in 10 (sample: resin) ratios, after combining 1-2 hour at 4 DEG C, by mixing for supernatant and nickel affine resin
It closes object to be transferred in chromatographic column, gravity stream elution discards efflux.Then cleaning buffer solution is added three times into chromatographic column
(100mM disodium hydrogen phosphates, 10mM trishydroxymethylaminomethanes, 8M urea, 20mM imidazoles, pH=8.0), cleaning buffer solution are overall
Product is 1 with resin volume ratio:8-1:10.After cleaning, then divide 2-3 elution albumen (100mM phosphorus from resin with eluent
Sour disodium hydrogen, 10mM trishydroxymethylaminomethanes, 8M urea, 20mM imidazoles, pH=4.5), amount and the tree of eluent are added every time
Fat is isometric.Purification of samples analyzes sample purity with dodecyl sodium sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).Egg
The sample buffer for being previously added 5% beta -mercaptoethanol is needed in white sample, and is heated 10 minutes at 95 DEG C, protein sample adds
It is downloaded to previously prepared 12.5%SDS-PAGE, the albumen volume containing the sample of each swimming lane is within the scope of 10-20ug.Vertical electrophoresis voltage
40-70min is run in 90-140V.Electrophoretic buffer contains 25mM trishydroxymethylaminomethanes, 250mM glycine, and 0.1% ten
Sodium dialkyl sulfate.With coomassie G-250 dyeing post-processing observation pillar locations after electrophoresis.
The obtained AXB and BXA albumen of purifying carries out digestion with TEV protease respectively, and digestion temperature is 0 DEG C or 37 DEG C,
The digestion time is 1h.
Embodiment 5：Size exclusion chromatography purifies
The Protein elution liquid of 500 μ L passes through on AKTA protein purification systems (AKTA Avant, GE Healthcare)
Gel permeation column Superdex 200increas 10/300GL carry out consummate.Mobile phase is PBS, flow velocity 0.5mL/min.It is logical
Cross monitoring A280Target peak is collected, and carries out SDS-PAGE analyses.The protein rope of high-purity can be obtained by step purifying
Hydrocarbon.The topological structure of purified product is partially digested and holoenzyme cuts confirmation by TEV.Fig. 2 and Fig. 6 shows that SEC is isolated and purified
AEXEB and EAXEB protein catennes as a result, the resid vol of wherein main peak is about 10.5mL, be target product protein rope
Hydrocarbon.Fig. 4 and Fig. 8 respectively illustrate that AEXEB and EAXEB protein catennes are partially digested and the result of whole digestions.Fig. 4 and figure
In 8,1 represents protein catenne, and 2,3 represent in various degree partially digested as a result, 4 represent that catenne holoenzyme is cut as a result, 5 represent
Cyclic products compare, 6 represent cyclic products it is partially digested as a result, 7 represent it is that cyclic products holoenzyme is cut as a result, 8 be linear product
Control.
The molecular weight of purified product is measured with MALDI _ TOFMS (MALDI-TOF), institute
It is 5800MALDI-TOF/TOF analyzers (AB SCIEX) with instrument.Fig. 3 and Fig. 7 shows prepared AEXEB and EAXEB
Protein catenne actual molecular weight is consistent with theoretical molecular weight.
Fig. 9 shows that AXB and BXA protein catennes be partially digested and the result of whole digestions.In Fig. 9, A-X-B and
B-X-A respectively represents corresponding protein catenne, and 37 DEG C of 0 DEG C of+TEV and+TEV respectively represent partially digested and complete degestion
Result.
Figure 10 shows that the actual molecular weight of the AXB and BXA of preparation are consistent with expected molecular amount.
Claims (6)
1. a kind of preparation method of protein catenne, includes the following steps：
1) protein catenne sequence monomer is designed, which includes at least the albumen being made of two polypeptide fragments
Qualitative response pair and the albumen interlocking structure unit between two polypeptide fragments, wherein the albumen interlocking structure unit makes
Mutual winding between molecule occurs for protein catenne monomer, and the albumen qualitative response is to making protein catenne monomer be cyclized
Form catenne；Specifically, the protein catenne monomer is based on following basic structure (a) or (b)：(a) successively from N-terminal to C-terminal
For spy label, tumor suppressor p53 oligomerization domains, spy hunter；(b) spy hunter, tumour suppression are followed successively by from N-terminal to C-terminal
Factor p53 oligomerization domains processed, spy label；It is few in spy label and tumor suppressor p53 at (a) or (b) on architecture basics
Between multimerisation domain, and/or, between spy hunter and tumor suppressor p53 oligomerization domains, and/or, at (a) or
(b) one or more arbitrary identical or different functional protein structural domain is added in the N-terminal of structure and/or C-terminal；
2) it builds the corresponding encoding gene of protein catenne monomer and is introduced into vector plasmid；
3) gene of step 2) structure is expressed in cell；
4) expression albumen is purified, obtains protein catenne.
2. preparation method as described in claim 1, which is characterized in that in the amino acid sequence of the spy label such as sequence table
SEQ ID No：Shown in 1, SEQ ID No in the amino acid sequence of the spy hunter such as sequence table：Shown in 3.
3. preparation method as described in claim 1, which is characterized in that the oligomerization domain of the tumor suppressor p53
Amino acid sequence such as sequence table in SEQ ID No：Shown in 5.
4. preparation method as described in claim 1, which is characterized in that the functional protein structural domain is that elastin laminin is similar
Object, red fluorescent protein, beta-lactamase and/or dihyrofolate reductase.
5. preparation method as described in claim 1, which is characterized in that the structure of the protein catenne monomer is from N-terminal to C-terminal
It is one of having structure：Spy label-tumor suppressor p53 oligomerization domains-spy hunter, spy label-functional protein structure
Domain-tumor suppressor p53 oligomerization domains-spy hunter, spy label-tumor suppressor p53 oligomerization domains-function
Protein structure domain-spy hunter, the-the first functional protein of spy label structural domain-tumor suppressor p53 oligomerization domains-the second
Functional protein structural domain-spy hunter, functional protein structural domain-spy label-tumor suppressor p53 oligomerization domains-spy are caught
Hand, spy label-tumor suppressor p53 oligomerization domains-spy hunter-functional protein structural domain, the first functional protein structure
Domain-spy label-tumor suppressor p53 oligomerization domains-spy hunter the-the second functional protein structural domain；It is either above-mentioned each
One of the structure that spy label and spy catcher's position obtain after exchanging in kind structure；The wherein described first functional protein structural domain and
Two functional protein structural domains are identical or different.
6. preparation method as described in claim 1, which is characterized in that His labels are added on protein catenne monomer, in step
It is rapid 4) to be purified by nickel affinity chromatography.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201510593863.9A CN105061581B (en) | 2015-09-17 | 2015-09-17 | Can gene code holoprotein catenne preparation method |
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| CN106591345A (en) * | 2016-12-26 | 2017-04-26 | 华侨大学 | Method for separation and purification and immobilization integration of recombinant double enzyme |
| CN107299107A (en) * | 2017-07-31 | 2017-10-27 | 北京大学 | A kind of preparation method of protein assembling functional material layer by layer |
| CN111073925B (en) * | 2018-10-19 | 2022-04-26 | 北京大学 | High-efficiency polypeptide-polypeptide coupling system and method based on disordered protein coupling enzyme |
| CN110272913B (en) * | 2019-06-12 | 2020-11-03 | 北京大学 | Protein coupling method based on soyabean |
| CN113045633B (en) * | 2019-12-27 | 2022-04-26 | 北京大学 | Design of protein heterogeneous entanglement primitive and preparation method of complex catenane structure |
| CN111560391B (en) * | 2020-05-21 | 2022-02-11 | 北京大学 | Biosynthesis method of protein heterogeneous catenane |
| CN112210019A (en) * | 2020-10-15 | 2021-01-12 | 广东省科学院生物工程研究所 | Purification method of transmembrane region of membrane protein containing single transmembrane region |
| CN113214369B (en) * | 2021-05-12 | 2022-05-17 | 南京工业大学 | Molecular peptide mutant |
| CN114075298B (en) * | 2022-01-07 | 2022-04-29 | 广州中科蓝华生物科技有限公司 | Soxhydrogenated VAR2CSA recombinant protein and preparation method and application thereof |
| CN117003852A (en) * | 2022-07-07 | 2023-11-07 | 北京大学 | Topology modification of interleukin-2 and its application as autoimmune disease medicine |
| CN116621947B (en) * | 2023-07-18 | 2023-11-07 | 北京智源人工智能研究院 | Topological protein based on Soxhlet skeleton, preparation method and application |
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