CN107299107A - A kind of preparation method of protein assembling functional material layer by layer - Google Patents
A kind of preparation method of protein assembling functional material layer by layer Download PDFInfo
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- CN107299107A CN107299107A CN201710637787.6A CN201710637787A CN107299107A CN 107299107 A CN107299107 A CN 107299107A CN 201710637787 A CN201710637787 A CN 201710637787A CN 107299107 A CN107299107 A CN 107299107A
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- 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
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Abstract
The invention discloses a kind of preparation method of protein assembling functional material layer by layer.First design protein layer assembly construction unit, the construction unit be the fusion protein comprising orthogonal albumen qualitative response pair and functional protein domain, wherein same protein reaction to two protein respectively be located at different construction units in；The corresponding gene of construction unit is introduced into expression vector, and expressed in cell；The protein of expression is purified, using orthogonal albumen qualitative response to carrying out layer assembly to protein, required function material is obtained.The form that the inventive method is expressed using gene code cell obtains high-purity and stable reactive protein, prepared material has interlayer covalent cross-linking characteristic, suitable for a variety of organic and inorganic material interfaces, and the bioactivity and response of wherein functional protein can be kept during modification and after modification.
Description
Technical field
The present invention relates to the preparation of large biological molecule, more particularly to the preparation method of protein and its functional assembly material and
Its surface and interface modification in application.
Background technology
It is highly important direction to carry out surface/interface to be modified using large biological molecule, because so can effectively carry
Its high biocompatibility, it might even be possible to assign its biological responding and Biofunctional.Wherein, protein is due to accurately may be used
The structure of control gets most of the attention with rich and varied function.
Laminated assembling technology is a kind of simple and reliable table/interface modification technology, and it can accurately control the composition of material
With structure.In recent years, this technology is widely used in various fields, including macromolecule, inorganic particulate, gold
Category etc. can be carried out effective assembling, and assign material special function.But in terms of biology, especially led in protein
In domain, due to the structure that protein is complicated, and the site that can be effectively reacted in unit-protein matter is less, therefore causes
It is difficult effectively to assemble.Simultaneously as the foldable structure and function of albumen are all very sensitive for chemical modification and environment, how
Ensure that the protein after assembling has the problem of stable structure and function are still one important.
At present, the report based on holoprotein layer assembly functional material is few, and one of example is exactly to utilize gene
Coding techniques prepares the elastin laminin analog with a large amount of opposite charges to (ELP), then using it each other
Electrostatic interaction carries out layer assembly, has prepared hollow microcapsules (Kolbe, A., et
al.Macromol.Rapid.Commun.2011,32,186-90.).By the end of current, not yet have been reported that proposition is prepared and had
Bioactivity and biological responding the holoprotein layer assembly material based on covalent effect.
The content of the invention
In order to prepare protein, component film is modified with the surface and interface for applying to material layer by layer, and the invention provides a kind of profit
With the method for recombinant protein orthogonal chemically reactive.
Protein interface layer assembly is realized, it is necessary to meet following condition：(1) need to set up in inorganic or gold
The bridge that category interface is connected with protein.(2) after alternately assembling process terminates each time, material surface should still have foot
Enough active reaction sites.For first condition, it is many it is specific chemical reaction as gold and sulfydryls between, silane coupler
Between silicon materials, have been obtained for being widely applied, or even had been developed that what a series of and inorganic material interacted
Particular polypeptide sequence etc..For Second Problem, someone using gene code tool design and prepared tool
Have a poly- polypeptide (ELP) of the elastic-like albumen of multiple opposite charges, this poly- polypeptide can on calcium carbonate layer assembly system
The biological capsule of standby core shell structure.But it is directed to that the problem of how material keeps bioactivity does not solve still.In recent years,
It developed series of biochemical reactions pair, such as spy hunter (SpyCatcher) and spy label (SpyTag) (Zakeri,
B., et al.Proc.Natl.Acad.Sci.USA 2012,109, E690-E697.), expedition hand (SnoopCatcher) and spy
Label (SnoopTag) (Veggiani, G.et al.M., Proc.Natl.Acad.Sci.USA 2016,113,1202-
1207.) etc..These reactions are to all from the protein instrument pair of micrococcus scarlatinae, general label segment is only ten
The small peptide of several amino acid, and hunter part is then one section of longer protein domain.They have preferable bio-compatible
Property, and efficient reaction can occur in physiological conditions.In addition, both verified reactions of previous work are complete between
It is complete orthogonal.If can the orthogonal albumen qualitative response of two kinds of reasonable design to the position in fusion protein and quantity, then will
The regeneration of the avtive spot in layer assembly can ideally be realized.The research of the present invention is found, by by both orthogonal eggs
White matter is reacted to being fused into new construction unit, layer assembly can be effectively realized, while may be incorporated into new functionalization
Domain, assigns the corresponding function of material.
Specifically, the technical scheme is that：
A kind of preparation method of protein assembling functional material layer by layer, comprises the following steps：
1) design protein layer assembly construction unit, the construction unit be comprising orthogonal albumen qualitative response pair, with
And the fusion protein of functional protein domain, wherein same protein reaction to two protein be located at different construct respectively
In unit；
2) build the gene corresponding to protein layer assembly construction unit and be introduced into expression vector, then in cell
The corresponding albumen of the constructed gene of expression；
3) protein of expression is purified, obtains high-purity protein layer assembly construction unit；
4) required function material is obtained to carrying out layer assembly to protein using orthogonal albumen qualitative response.
Above-mentioned steps 1) described in orthogonal albumen qualitative response select spy hunter and spy tag reactant pair to preferential, and
Expedition hand and spy tag reactant pair.
SEQ ID No in typical spy label (abbreviation A) and the amino acid sequence of spy hunter (abbreviation B) such as sequence table：1 He
SEQ ID No：Shown in 2, its corresponding gene order can be SEQ ID No in sequence table：3 and SEQ ID No：4, can also
It is other gene orders that the degeneracy based on codon encodes same amino acid sequence.
It is typical to visit SEQ ID No in label (abbreviation C) and the amino acid sequence of expedition hand (abbreviation B) such as sequence table：5 Hes
SEQ ID No：Shown in 6, its corresponding gene order can be SEQ ID No in sequence table：7 and SEQ ID No：8, and be based on
The gene order of the degeneracy of codon or the same amino acid sequence of other codings.
Certainly, other mutually orthogonal albumen qualitative responses are to that can also apply herein.
Can also include except orthogonal albumen qualitative response is in addition to, in protein construction unit one or more it is identical or
Different functional protein domains, these functional protein domains are located between orthogonal albumen qualitative response pair.The functional protein
Can be elastin laminin analog (ELP), nano antibody (nanobody), affine body (Affibody), super uranium associated proteins
(SUP), dihyrofolate reductase (DHFR) etc., and other functional proteins that medical science, agricultural, industry, scientific research field are related to.
Illustrate the structure of protein layer layer assembling construction unit below by some specific examples：
(a) label-spy hunter-spy label (CBC) is visited：It is respectively to visit label, spy hunter, visit label from N-terminal to C-terminal, its
In every section visit and be all inserted into an elastin laminin analog (ELP) between label and spy hunter, its corresponding gene order can be
SEQ ID No in sequence table：9, i.e. CBC, wherein 6-11 amino acids residue is 6 × His, 16-28 amino acids residues
To visit label, 30-108 amino acids residue is ELP, and 123-249 amino acids residue is spy hunter, 251-329
Amino acid residue is ELP, and 344-356 amino acids residue is spy label.
(b) expedition hand-spy label-expedition hand (DAD)：It is respectively expedition hand, spy label, expedition hand from N-terminal to C-terminal, its
In an elastin laminin analog (ELP) is all inserted between every section of expedition hand and spy label, its corresponding gene order can be
SEQ ID No in sequence table：10, i.e. DAD, wherein 6-11 amino acids residue is 6 × His, 16-128 amino acids are residual
Base is expedition hand, and 134-210 amino acids residue is ELP, and 213-225 amino acids residue is spy label, 227-305
Amino acids residue is ELP, and 320-432 amino acids residue is expedition hand.
In addition, CBC can also by wherein first ELP structure replacing into super uranium associated proteins structure (SUP) or
Dihyrofolate reductase (DHFR), its corresponding gene order can be SEQ ID No in sequence table：11, SEQ ID No：12
That is CBC-SUP, CBC-DHFR.In SEQ ID No：In 11,6-11 amino acids residue is 6 × His, 16-28 bit aminos
Sour residue is visits label, and 31-110 amino acids residue is SUP, and 112-238 amino acids residue is spy hunter, 252-
318 amino acids residues are ELP, and 333-345 amino acids residue is spy label.In SEQ ID No：In 12,6-11
Amino acid residue is 6 × His, and 16-28 amino acids residue is visits label, and 31-189 amino acids residue is DHFR, the
192-318 amino acids residue is spy hunter, and 321-395 amino acids residue is ELP, 413-425 amino acids residues
To visit label.
Above-mentioned steps 2) gene corresponding to protein layer assembly construction unit is built by recombinant DNA technology, and draw
Enter into suitable vector plasmid.The applicable carrier such as pQE and pET series plasmids of prokaryotic expression system.To be follow-up pure
It is convenient to change, and N-terminal adds 6 × His labels on the construction unit of protein layer assembly.Typically the plasmid of structure is imported big
The protein layer assembly construction unit is expressed in the engineering bacterias such as enterobacteria, expression temperature is general at 20 DEG C or so, during expression
Between generally higher than 12 hours.
Above-mentioned steps 3) in, conventional purification process is that list is constructed in the protein layer assembly for the addition of His labels
Member carries out nickel affinity chromatography purifying.
Above-mentioned steps 4) in, implementing the premise of interface layer level assembling needs a bridge between linkage interface and protein to make
Use site.By taking gold surface as an example, first layer group is carried out by the construction unit forerunner body protein containing multiple cysteine residues
Dress.The present invention devises a kind of spy label-elastin laminin analog-spy label-elastin laminin analog-Guang of spy label -4 × half
Forerunner's body protein of propylhomoserin (AAA-4Cys), its amino acid sequence can be SEQ ID No in sequence table：13, wherein：6-11
Amino acids residue is 6 × His；14-26 amino acids residue is spy label；31-108 amino acids residue is ELP；The
111-123 amino acids residue is spy label, and 125-203 amino acids residue is ELP；208-220 amino acids residues
For spy label；223-227 amino acids residue is 4 × cysteine.Cysteine meeting and gold surface under reduction-state
The effective specific reaction of generation, and spy label can be as next layer of reaction site with visiting label-spy hunter-spy label
(CBC) the spy hunter in albumen is reacted, because spy chemically reacts between spy chemical reaction in complete orthogonality, CBC
Remaining label of visiting can continue as active reaction sites and the expedition in next layer of expedition hand-spy label-expedition hand (DAD)
Hand is reacted.So the remaining active reactive group of material surface becomes spy label again, and so move in circles progress
The alternating action of label-spy hunter-spy label (CBC) and expedition hand-spy label-expedition hand (DAD) is visited, can be prepared with friendship
It is coupled the protein film of the layer assembly of structure.
In addition to gold surface, method of the invention applies also for silica-base material surface, it is possible to use amino silane is coupled
Agent carries out amino modified, modified recycling 3- maleimidopropionic acid n-hydroxysuccinimides to silicon materials surface in advance
Ester, makes its surface be rich in maleimide base group, so passes through above-mentioned spy label-elastin laminin analog-spy label-elasticity
Forerunner's body protein effect of albumen analog--4 × cysteine of spy label (AAA-4Cys) can also realize protein group layer by layer
Dress.This method is also applicable to the surfaces such as mica, glass.
It is noted that above-mentioned preparation method, which is applicable not only to two dimensional surface substrate, can also be applied to three-dimensional stand
Body material.
The protein material assembled can carry out functional test, in some embodiments of the invention, from CBC-SUP
Enrichment and the release experiment of uranyl cation are carried out, and CBC-DHFR is to the reduction experiment of dihydrofoilic acid.
The technical advantage of the protein layer assembly construction unit preparation method of the present invention is essentially consisted in：Gene can be utilized
The form of coding, obtains that purity is higher and reactive protein of function-stable；And the film that layer assembly is prepared has interlayer
Covalent cross-linking characteristic, and during modification and rear film can keep the wherein bioactivity of functional protein and response
Property；In addition, assemble method is applied to a variety of organic and inorganic material interfaces, such as metal, silicon material to protein of the invention layer by layer
Material etc..
Brief description of the drawings
Fig. 1 shows fusion protein CBC, DAD and CBC-SUP purifying and the reaction feelings between them in embodiment 2
Condition.
Fig. 2 shows that fusion protein CBC, DAD and AAA-4Cys purifying and DAD and AAA-4Cys divide in embodiment 2
Not with CBC response situation.
Fig. 3 shows the purifying of fusion protein CBC-DHFR and CBC-Affibody in embodiment 3, and they with DAD,
Response situation between AAA-4Cys.
Fig. 4 shows CBC, DAD and CBC-SUP circular dichroism spectra data.
Fig. 5 shows CBC, DAD and CBC-SUP size exclusion chromatograph data.
Fig. 6 shows CBC (a), DAD (b), CBC-SUP (c) flight time mass spectrum data and CBC-DHFR (d) with
And AAA-4Cys (e) High Performance Liquid Chromatography-Electrospray Ionization Mass Spectrometry data.
Fig. 7 shows the CBC/DAD (AAA-4Cys) (a) and CBC- under the detection of microcrystalline quartz balance in embodiment 4
SUP/DAD (AAA-4Cys) (b) layer assembly data.
Fig. 8 shows enrichment and release experiment of the CBC-SUP/DAD layer assemblies material in uranyl cation in embodiment 5
Photo：Wherein, SG is unmodified silica gel；SG-NH2The silica gel (silane coupler) being modified for surface amination；SG-MAL is
The silica gel (3- maleimidopropionic acids N-hydroxy-succinamide ester) that surface maleimation is modified；SG-SUP is surface group
The silica gel (a) and detection data (b) of CBC-SUP multilayer protein films are filled.
Fig. 9 shows the standard curve (a) containing NADPH absorbances under conditions of protein silica gel in embodiment 6, and
Dynamics data (b) of the CBC-DHFR/DAD layer assemblies material in DHF reduction experiments.
Embodiment
Below by embodiment, further the present invention is described in detail, the model of but do not limit the invention in any way
Enclose.
Prepare concretely comprising the following steps for protein layer assembly construction unit：Built and contained by recombination engineering technology
His6Label (be used for protein purification), visit label (C), elastin laminin analog (E) (or SUP, DHFR and Affibody
Deng other albumen), spy hunter (B), elastin laminin analog (E), visit label (C) fusion protein, i.e. C (E) B (E) C gene
Sequence is simultaneously inserted in expression vector；It is similar with expedition hand (D), elastin laminin analog (E), spy label (A), elastin laminin is built
The fusion protein of thing (E), expedition hand (D), i.e. D (E) A (E) D gene order is simultaneously inserted in expression vector；Then molecule is passed through
Expression vector is transformed into expression in escherichia coli fusion protein by cloning process, then is obtained by the protein purification method such as affinity chromatography
The subject fusion proteins that must be purified.The subject fusion proteins are for example：CBC、DAD、CBC-SUP、CBC-DHFR、AAA-4cys
Deng.
Utilize sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), size exclusion chromatography, Matrix-assisted
The means such as laser desorption ionisation flight time mass spectrum characterize the properties such as the molecular weight of protein construction unit, topological structure；And
And carried out in-situ test using microcrystalline quartz balance and assembling of the dissipation instrument to film；It is thin to protein using analysis method
The function of membrane material makes assessment.
Embodiment 1：The conversion and expression of protein construction unit target gene
Above protein sequence is built in pQE-80L expression vectors, is then expressed in e. coli bl21 (DE3).
Before extensive expression, it (is 100 μ g/ containing concentration that the monoclonal bacterium for first obtaining conversion, which is seeded in 5mL 2XYT culture mediums,
ML ampicillins), at 37 DEG C, concussion and cultivate is stayed overnight under the conditions of 250rpm, is transferred within second day the fresh 2 × YT cultures of 1L
(it is placed in 3L triangular flask, be 100 μ g/mL ampicillins containing concentration), at 37 DEG C, is advised greatly under the conditions of 250rpm in base
The culture of mould, treats its OD600Grow between 0.6-1.0, add final concentration of 0.1mM isopropyl-β-D-thiogalactoside
(IPTG) induce, then cultivation temperature is set to 22 DEG C, and 220rpm collects thalline after cultivating 20h.
Embodiment 2：The purifying of protein construction unit
1L E. coli broths are collected in centrifugal bottle, thalline is collected with 5000g centrifugal forces, upper strata culture is removed
Liquid.Thalline is resuspended with 25mL denaturation cushioning liquid A (50mM disodium hydrogen phosphates, 300mM sodium chloride, 10mM imidazoles, pH=8.0),
Ultrasound Instrument smudge cells under the conditions of 4 DEG C is used again, then centrifuges Escherichia coli breakdown products under 4 DEG C, 28000g centrifugal force
20 minutes.Obtained supernatant is mixed with being denatured the nickel affine resin of cushioning liquid A balances, and 1h is stirred at 4 DEG C.Mixture
Be placed in void column, carry out gravity stream elution, eluent for denaturation cushioning liquid B (50mM disodium hydrogen phosphates, 300mM sodium chloride,
300mM imidazoles, pH=8.0).Protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- obtained by elution
PAGE purity) is tested.Analysis sample is mixed with 5 × protein sample-loading buffer, after 98 DEG C of heating 10min, is loaded into 1,0%0
In sodium dialkyl sulfate-polyacrylamide gel, 70min (electrophoresis liquids are run under vertical electrophoresis 80-140V voltages：The hydroxyls of 25mM tri-
Base aminomethane, 250mM glycine, 0.1% lauryl sodium sulfate).Gel is observed after being handled with Coomassie brilliant blue G-250 dyeing
Pillar location.Fig. 1 and Fig. 2 shows that the nickel of CBC, DAD, CBC-SUP and AAA-4Cys albumen at different temperatures is affine
Chromatographic purifying situation, by Bacillus coli expression, the purifying of nickel affinity chromatography post can obtain the protein layer assembly of higher degree
Construction unit, while the reactivity between orthogonality and protein between showing CBC/DAD.
Embodiment 3：The sign of protein construction unit
500 μ L Protein elution liquid passes through on AKTA protein purification systems (AKTA Avant, GE Healthcare)
Gel permeation column Superdex 200Increase 10/300GL carry out consummate.Mobile phase is PBS, and flow velocity is 0.5mL/min.
By monitoring A280Target peak, SDS-PAGE electrophoresis results are collected as shown in figure 3, then protein solution is carried out except salt treatment is used for
Circular dichroism spectra (CD) analysis is carried out, as a result as shown in Figure 4.
The molecular weight of purified product is with MALDI _ TOFMS (MALDI-TOF) or efficiently
Chromatography-electrospray-ionization/mass spectrometry is determined, and instrument is 5800MALDI-TOF/TOF analyzers (AB SCIEX).Fig. 5 is shown
Prepared protein layer assembly construction unit actual molecular weight and theoretical molecular is basically identical.CBC、DAD、CBC-
SUP, CBC-DHFR and AAA-4Cys mass spectrometric data are as shown in Figure 6.
Embodiment 4：The preparation of protein component film layer by layer
Above-mentioned albumen is replaced into cushioning liquid into phosphate-buffered liquid system (PBS, 50mM disodium hydrogen phosphate and biphosphate
Sodium, pH7.4), 1mM TCEP are added in AAA-4Cys to be used to protect sulfydryl.Here by taking the silica gel particle being modified as an example, enter
Row operations described below：
1) it is firstly added in the modified silica-gel system that 1mL 0.5mg/mL AAA-4Cys is 0.5mL to volume, vibration
After 20min, 5000rpm centrifugations 1min.1ml PBSs are rejoined after removing supernatant, for cleaning.Vibrate after 1min
5000rpm centrifuges 1min again, removes supernatant.
2) and then 1mL 0.5mg/mL CBC either CBC-SUP PBS solutions are added, repeats aforesaid operations until clear
Operation is washed to terminate.
3) PBS solution that 1mL 0.5mg/mL DAD is added after repeats aforesaid operations until cleaning operation terminates.
Such a CBC/DAD double-layer assembled has been completed, be repeatedly sequentially repeated 2), 3) operation can obtain multilayer
Protein package assembly layer by layer.
By taking gold surface as an example, operations described below is carried out：
1) 0.5mg/mL AAA-4Cys solution immersion 20min is added, then 10min is embathed using PBS.
2) 0.5mg/mL CBC solution immersion 20min is added, then 10min is embathed using PBS.
3) 0.5mg/mL DAD solution immersion 20min is added, then 10min is embathed using PBS.
Such a CBC/DAD double-layer assembled has been completed, be repeatedly sequentially repeated 2), 3) operation can obtain multilayer
Protein component film layer by layer.Monitored it is equally possible that carrying out layer assembly in situ to gold surface using microcrystalline quartz balance,
Concrete operations are similar to gold surface infusion process, as a result as shown in Figure 7.
Embodiment 5：Protein layer by layer test by the uranium enrichment of component film and release function
CBC-SUP is prepared by aforesaid operations, and is carried out according to the method for embodiment 4 on modified silica-gel surface with DAD
0.5mLl protein resin is added in chromatographic column by layer assembly preparation after finishing, and be passed through 200mL contains 10nM uranyl
The ultra-pure water solution of cation；The sal volatile for first adding 300 μ L50mM (can be suitably pressurizeed) after being passed through and finishing (now with existing
With) stand after 10min or so, then allow it to flow out naturally；Then again in three times, the sal volatile of same volume is sequentially added,
The solution of all outflows is collected in case detection；After outflow terminates, the ultra-pure water for adding certain volume is washed.Repeat above-mentioned
Operation 10 acquisitions, 10 testing samples.
The method that testing sample tries out arsenazo III is monitored, and the sample of the normal concentration of various concentrations is prepared first
The drafting of standard curve is carried out, after drafting is finished, testing sample adds 80 μM of azos for 0.1M hydrochloric acid containing concentration in equal volume
Arsine III solution.The absorbance at 652nm is tested using ELIASA, obtained data are converted into uranyl with standard curve
The concentration of cation, specific data are as shown in Figure 8.
Embodiment 6：Protein layer by layer component film dihydrofolate reduction test
It is same as Example 5, CBC-DHFR is prepared, is carried out on modified silica-gel surface with DAD after layer assembly, takes 1 μ
L resins are added to (50mM phosphate, 80%K in 184 μ L buffer solutions2HPO4, 20%KH2PO4, 1mM mercaptoethanols) and then add
5 μ L 20mM NADPHs (NADPH, Sigma) and 10 μ L 5mM dihydrofoilic acid (DHF,
Sigma).Used after mixing in the range of ELIASA kinetics model measurement certain time (>30min) absorbance change at 340nm.
Before the experiments, the Specification Curve of Increasing of NADPH absorbances at 340nm of the various concentrations containing resin should be carried out.According to
The quality that standard curve is converted into NADPH consumption divided by resin within the unit interval can obtain the enzymatic activity of resin, number
According to as shown in Figure 9.
SEQUENCE LISTING
<110>Peking University
<120>A kind of preparation method of protein assembling functional material layer by layer
<130> WX2017-03-112
<160> 13
<170> PatentIn version 3.3
<210> 1
<211> 13
<212> PRT
<213>Artificial sequence
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Ala His Ile Val Met Val Asp Ala Tyr Lys Pro Thr Lys
1 5 10
<210> 2
<211> 127
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<213>Artificial sequence
<400> 2
Ile Pro Thr Thr Glu Asn Leu Tyr Phe Gln Gly Ala Met Val Asp Thr
1 5 10 15
Leu Ser Gly Leu Ser Ser Glu Gln Gly Gln Ser Gly Asp Met Thr Ile
20 25 30
Glu Glu Asp Ser Ala Thr His Ile Lys Phe Ser Lys Arg Asp Glu Asp
35 40 45
Gly Lys Glu Leu Ala Gly Ala Thr Met Glu Leu Arg Asp Ser Ser Gly
50 55 60
Lys Thr Ile Ser Thr Trp Ile Ser Asp Gly Gln Val Lys Asp Phe Tyr
65 70 75 80
Leu Tyr Pro Gly Lys Tyr Thr Phe Val Glu Thr Ala Ala Pro Asp Gly
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Tyr Glu Val Ala Thr Ala Ile Thr Phe Thr Val Asn Glu Gln Gly Gln
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Val Thr Val Asn Gly Lys Ala Thr Lys Gly Asp Ala His Ile Asp
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gcccatattg tcatggttga tgcatacaag ccgacgaag 39
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atcccaacga ccgaaaacct gtattttcag ggcgccatgg ttgatacctt atcaggttta 60
tcaagtgagc aaggtcagtc cggtgatatg acaattgaag aagatagtgc tacccatatt 120
aaattctcaa aacgtgatga ggacggcaaa gagttagctg gtgcaactat ggagttgcgt 180
gattcatctg gtaaaactat tagtacatgg atttcagatg gacaagtgaa agatttctac 240
ctgtatccag gaaaatatac atttgtcgaa accgcagcac cagacggtta tgaggtagca 300
actgctatta cctttacagt taatgagcaa ggtcaggtta ctgtaaatgg caaagcaact 360
aaaggtgacg ctcatattga c 381
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Asp Tyr Pro Asp Ile Tyr Gly Ala Ile Asp Gln Asn Gly Thr Tyr Gln
20 25 30
Asn Val Arg Thr Gly Glu Asp Gly Lys Leu Thr Phe Lys Asn Leu Ser
35 40 45
Asp Gly Lys Tyr Arg Leu Phe Glu Asn Ser Glu Pro Ala Gly Tyr Lys
50 55 60
Pro Val Gln Asn Lys Pro Ile Val Ala Phe Gln Ile Val Asn Gly Glu
65 70 75 80
Val Arg Asp Val Thr Ser Ile Val Pro Gln Asp Ile Pro Ala Thr Tyr
85 90 95
Glu Phe Thr Asn Gly Lys His Tyr Ile Thr Asn Glu Pro Ile Pro Pro
100 105 110
Lys
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ggaaaactgg gcgatattga atttattaaa gtgaacaaa 39
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atgaagccgc tgcgtggtgc cgtgtttagc ctgcagaaac agcatcccga ctatcccgat 60
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aaactgacct ttaagaatct gagcgatggc aaatatcgcc tgtttgaaaa tagcgaaccc 180
gctggctata aaccggtgca gaataagccg attgtggcgt ttcagattgt gaatggcgaa 240
gtgcgtgatg tgaccagcat tgtgccgcag gatattccgg ctacatatga atttaccaac 300
ggtaaacatt atatcaccaa tgaaccgata ccgccgaaa 339
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Met Lys Gly Ser Ser His His His His His His Val Glu Ala Ser Gly
1 5 10 15
Lys Leu Gly Asp Ile Glu Phe Ile Lys Val Asn Lys Val Asp Val Pro
20 25 30
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Glu Gly Val Pro Gly
35 40 45
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
50 55 60
Gly Val Pro Gly Glu Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
65 70 75 80
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Glu Gly Val
85 90 95
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Gly Leu Leu Asp
100 105 110
Gly Pro Gln Gly Ile Trp Gly Gln Glu Leu Ile Pro Thr Thr Glu Asn
115 120 125
Leu Tyr Phe Gln Gly Ala Met Val Asp Thr Leu Ser Gly Leu Ser Ser
130 135 140
Glu Gln Gly Gln Ser Gly Asp Met Thr Ile Glu Glu Asp Ser Ala Thr
145 150 155 160
His Ile Lys Phe Ser Lys Arg Asp Glu Asp Gly Lys Glu Leu Ala Gly
165 170 175
Ala Thr Met Glu Leu Arg Asp Ser Ser Gly Lys Thr Ile Ser Thr Trp
180 185 190
Ile Ser Asp Gly Gln Val Lys Asp Phe Tyr Leu Tyr Pro Gly Lys Tyr
195 200 205
Thr Phe Val Glu Thr Ala Ala Pro Asp Gly Tyr Glu Val Ala Thr Ala
210 215 220
Ile Thr Phe Thr Val Asn Glu Gln Gly Gln Val Thr Val Asn Gly Lys
225 230 235 240
Ala Thr Lys Gly Asp Ala His Ile Asp Thr Ser Val Pro Gly Val Gly
245 250 255
Val Pro Gly Val Gly Val Pro Gly Glu Gly Val Pro Gly Val Gly Val
260 265 270
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
275 280 285
Gly Glu Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
290 295 300
Val Gly Val Pro Gly Val Gly Val Pro Gly Glu Gly Val Pro Gly Val
305 310 315 320
Gly Val Pro Gly Val Gly Val Pro Gly Gly Leu Leu Asp Gly Pro Gln
325 330 335
Gly Ile Trp Gly Gln Leu Glu Gly Lys Leu Gly Asp Ile Glu Phe Ile
340 345 350
Lys Val Asn Lys Gly Thr Val Glu Lys Lys Met
355 360
<210> 10
<211> 439
<212> PRT
<213>Artificial sequence
<400> 10
Met Lys Gly Ser Ser His His His His His His Val Glu Ala Ser Met
1 5 10 15
Lys Pro Leu Arg Gly Ala Val Phe Ser Leu Gln Lys Gln His Pro Asp
20 25 30
Tyr Pro Asp Ile Tyr Gly Ala Ile Asp Gln Asn Gly Thr Tyr Gln Asn
35 40 45
Val Arg Thr Gly Glu Asp Gly Lys Leu Thr Phe Lys Asn Leu Ser Asp
50 55 60
Gly Lys Tyr Arg Leu Phe Glu Asn Ser Glu Pro Ala Gly Tyr Lys Pro
65 70 75 80
Val Gln Asn Lys Pro Ile Val Ala Phe Gln Ile Val Asn Gly Glu Val
85 90 95
Arg Asp Val Thr Ser Ile Val Pro Gln Asp Ile Pro Ala Thr Tyr Glu
100 105 110
Phe Thr Asn Gly Lys His Tyr Ile Thr Asn Glu Pro Ile Pro Pro Lys
115 120 125
Val Asp Gly His Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
130 135 140
Gly Val Pro Gly Glu Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
145 150 155 160
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Glu Gly Val
165 170 175
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
180 185 190
Gly Val Gly Val Pro Gly Glu Gly Val Pro Gly Val Gly Val Pro Gly
195 200 205
Val Gly Glu Leu Ala His Ile Val Met Val Asp Ala Tyr Lys Pro Thr
210 215 220
Lys Thr Ser Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
225 230 235 240
Glu Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
245 250 255
Gly Val Pro Gly Val Gly Val Pro Gly Glu Gly Val Pro Gly Val Gly
260 265 270
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
275 280 285
Pro Gly Glu Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
290 295 300
Gly Gly Leu Leu Asp Gly Pro Gln Gly Ile Trp Gly Gln Leu Glu Met
305 310 315 320
Lys Pro Leu Arg Gly Ala Val Phe Ser Leu Gln Lys Gln His Pro Asp
325 330 335
Tyr Pro Asp Ile Tyr Gly Ala Ile Asp Gln Asn Gly Thr Tyr Gln Asn
340 345 350
Val Arg Thr Gly Glu Asp Gly Lys Leu Thr Phe Lys Asn Leu Ser Asp
355 360 365
Gly Lys Tyr Arg Leu Phe Glu Asn Ser Glu Pro Ala Gly Tyr Lys Pro
370 375 380
Val Gln Asn Lys Pro Ile Val Ala Phe Gln Ile Val Asn Gly Glu Val
385 390 395 400
Arg Asp Val Thr Ser Ile Val Pro Gln Asp Ile Pro Ala Thr Tyr Glu
405 410 415
Phe Thr Asn Gly Lys His Tyr Ile Thr Asn Glu Pro Ile Pro Pro Lys
420 425 430
Gly Thr Val Glu Lys Lys Met
435
<210> 11
<211> 352
<212> PRT
<213>Artificial sequence
<400> 11
Met Lys Gly Ser Ser His His His His His His Val Glu Ala Ser Gly
1 5 10 15
Lys Leu Gly Asp Ile Glu Phe Ile Lys Val Asn Lys Val Asp Leu Asp
20 25 30
Cys Arg Glu Arg Ile Glu Lys Asp Leu Glu Asp Leu Glu Lys Glu Leu
35 40 45
Met Glu Met Lys Ser Ile Lys Leu Ser Asp Asp Glu Glu Ala Val Val
50 55 60
Glu Arg Ala Leu Asn Tyr Arg Asp Asp Ser Val Tyr Tyr Leu Glu Lys
65 70 75 80
Gly Asp His Ile Thr Ser Phe Gly Cys Ile Thr Tyr Ala Glu Gly Leu
85 90 95
Thr Asp Ser Leu Arg Met Leu His Arg Ile Ile Glu Gly Glu Leu Ile
100 105 110
Pro Thr Thr Glu Asn Leu Tyr Phe Gln Gly Ala Met Val Asp Thr Leu
115 120 125
Ser Gly Leu Ser Ser Glu Gln Gly Gln Ser Gly Asp Met Thr Ile Glu
130 135 140
Glu Asp Ser Ala Thr His Ile Lys Phe Ser Lys Arg Asp Glu Asp Gly
145 150 155 160
Lys Glu Leu Ala Gly Ala Thr Met Glu Leu Arg Asp Ser Ser Gly Lys
165 170 175
Thr Ile Ser Thr Trp Ile Ser Asp Gly Gln Val Lys Asp Phe Tyr Leu
180 185 190
Tyr Pro Gly Lys Tyr Thr Phe Val Glu Thr Ala Ala Pro Asp Gly Tyr
195 200 205
Glu Val Ala Thr Ala Ile Thr Phe Thr Val Asn Glu Gln Gly Gln Val
210 215 220
Thr Val Asn Gly Lys Ala Thr Lys Gly Asp Ala His Ile Asp Thr Ser
225 230 235 240
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Glu Gly Val
245 250 255
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
260 265 270
Gly Val Gly Val Pro Gly Glu Gly Val Pro Gly Val Gly Val Pro Gly
275 280 285
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Glu
290 295 300
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Gly Leu
305 310 315 320
Leu Asp Gly Pro Gln Gly Ile Trp Gly Gln Leu Glu Gly Lys Leu Gly
325 330 335
Asp Ile Glu Phe Ile Lys Val Asn Lys Gly Thr Val Glu Lys Lys Met
340 345 350
<210> 12
<211> 432
<212> PRT
<213>Artificial sequence
<400> 12
Met Lys Gly Ser Ser His His His His His His Val Glu Ala Ser Gly
1 5 10 15
Lys Leu Gly Asp Ile Glu Phe Ile Lys Val Asn Lys Val Asp Met Ile
20 25 30
Ser Leu Ile Ala Ala Leu Ala Val Asp Arg Val Ile Gly Met Glu Asn
35 40 45
Ala Met Pro Trp Asn Leu Pro Ala Asp Leu Ala Trp Phe Lys Arg Asn
50 55 60
Thr Leu Asn Lys Pro Val Ile Met Gly Arg His Thr Trp Glu Ser Ile
65 70 75 80
Gly Arg Pro Leu Pro Gly Arg Lys Asn Ile Ile Leu Ser Ser Gln Pro
85 90 95
Gly Thr Asp Asp Arg Val Thr Trp Val Lys Ser Val Asp Glu Ala Ile
100 105 110
Ala Ala Cys Gly Asp Val Pro Glu Ile Met Val Ile Gly Gly Gly Arg
115 120 125
Val Tyr Glu Gln Phe Leu Pro Lys Ala Gln Lys Leu Tyr Leu Thr His
130 135 140
Ile Asp Ala Glu Val Glu Gly Asp Thr His Phe Pro Asp Tyr Glu Pro
145 150 155 160
Asp Asp Trp Glu Ser Val Phe Ser Glu Phe His Asp Ala Asp Ala Gln
165 170 175
Asn Ser His Ser Tyr Cys Phe Glu Ile Leu Glu Arg Arg Glu Leu Ile
180 185 190
Pro Thr Thr Glu Asn Leu Tyr Phe Gln Gly Ala Met Val Asp Thr Leu
195 200 205
Ser Gly Leu Ser Ser Glu Gln Gly Gln Ser Gly Asp Met Thr Ile Glu
210 215 220
Glu Asp Ser Ala Thr His Ile Lys Phe Ser Lys Arg Asp Glu Asp Gly
225 230 235 240
Lys Glu Leu Ala Gly Ala Thr Met Glu Leu Arg Asp Ser Ser Gly Lys
245 250 255
Thr Ile Ser Thr Trp Ile Ser Asp Gly Gln Val Lys Asp Phe Tyr Leu
260 265 270
Tyr Pro Gly Lys Tyr Thr Phe Val Glu Thr Ala Ala Pro Asp Gly Tyr
275 280 285
Glu Val Ala Thr Ala Ile Thr Phe Thr Val Asn Glu Gln Gly Gln Val
290 295 300
Thr Val Asn Gly Lys Ala Thr Lys Gly Asp Ala His Ile Asp Thr Ser
305 310 315 320
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Glu Gly Val
325 330 335
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
340 345 350
Gly Val Gly Val Pro Gly Glu Gly Val Pro Gly Val Gly Val Pro Gly
355 360 365
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Glu
370 375 380
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Gly Leu
385 390 395 400
Leu Asp Gly Pro Gln Gly Ile Trp Gly Gln Leu Glu Gly Lys Leu Gly
405 410 415
Asp Ile Glu Phe Ile Lys Val Asn Lys Gly Thr Val Glu Lys Lys Met
420 425 430
<210> 13
<211> 229
<212> PRT
<213>Artificial sequence
<400> 13
Met Lys Gly Ser Ser His His His His His His Val Asp Ala His Ile
1 5 10 15
Val Met Val Asp Ala Tyr Lys Pro Thr Lys Leu Asp Gly His Gly Val
20 25 30
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Glu Gly
35 40 45
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
50 55 60
Pro Gly Val Gly Val Pro Gly Glu Gly Val Pro Gly Val Gly Val Pro
65 70 75 80
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
85 90 95
Glu Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Glu Leu Ala His
100 105 110
Ile Val Met Val Asp Ala Tyr Lys Pro Thr Lys Thr Ser Val Pro Gly
115 120 125
Val Gly Val Pro Gly Val Gly Val Pro Gly Glu Gly Val Pro Gly Val
130 135 140
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
145 150 155 160
Val Pro Gly Glu Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
165 170 175
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Glu Gly Val Pro
180 185 190
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Gly Leu Leu Asp Ala
195 200 205
His Ile Val Met Val Asp Ala Tyr Lys Pro Thr Lys Leu Glu Cys Cys
210 215 220
Cys Cys Trp Lys Lys
225
Claims (10)
1. a kind of preparation method of protein assembling functional material layer by layer, comprises the following steps：
1) protein layer assembly construction unit is designed, the construction unit is to include orthogonal albumen qualitative response pair and function
The fusion protein of protein structure domain, wherein same protein reaction to two protein respectively be located at different construction units
It is interior；
2) build the gene corresponding to protein layer assembly construction unit and be introduced into expression vector, then expressed in cell
The corresponding albumen of constructed gene；
3) protein of expression is purified, obtains high-purity protein layer assembly construction unit；
4) required function material is obtained to carrying out layer assembly to protein using orthogonal albumen qualitative response.
2. preparation method as claimed in claim 1, it is characterised in that the orthogonal albumen qualitative response is to for spy hunter and spy
Tag reactant pair, and expedition hand and spy tag reactant pair, or other orthogonal albumen qualitative responses pair.
3. preparation method as claimed in claim 2, it is characterised in that the spy label and the amino acid sequence of spy hunter difference
Such as SEQ ID No in sequence table：1 and SEQ ID No：Shown in 2, the amino acid sequence of label and expedition hand is visited respectively such as sequence table
Middle SEQ ID No：5 and SEQ ID No：Shown in 6.
4. preparation method as claimed in claim 3, it is characterised in that the spy label and the corresponding gene order point of spy hunter
Not such as SEQ ID No in sequence table：3 and SEQ ID No：Shown in 4, the corresponding gene order of the spy label and expedition hand is distinguished
Such as SEQ ID No in sequence table：7 and SEQ ID No：Shown in 8.
5. preparation method as claimed in claim 1, it is characterised in that it is identical that the construction unit includes one or more
Or different functional protein domains, the functional protein domain is between orthogonal albumen qualitative response pair.
6. preparation method as claimed in claim 5, it is characterised in that it is similar that the functional protein domain is selected from elastin laminin
One or more in thing, nano antibody, affine body, super uranium associated proteins and dihyrofolate reductase.
7. preparation method as claimed in claim 1, it is characterised in that the protein layer assembly construction unit is comprising spy
The construction unit of label-spy hunter-spy label and the construction unit for including expedition hand-spy label-expedition hand, are visiting label and spy
Between hunter, and insertions function protein structure domain between expedition hand and spy label.
8. preparation method as claimed in claim 1, it is characterised in that the protein layer assembly construction unit has in N-terminal
There are 6 × His labels.
9. preparation method as claimed in claim 1, it is characterised in that step 4) protein is carried out on the interface of layer assembly
With the bridge being connected with protein.
10. preparation method as claimed in claim 9, it is characterised in that the interface of layer assembly is gold surface, by containing many
The construction unit forerunner body protein of individual cysteine residues carries out first layer assembling；Or, the interface of layer assembly is silicon substrate
Expect surface, amino modified, modified recycling 3- maleimides are carried out to silicon materials surface in advance using amino silicane coupling agent
Amine propionic acid N-hydroxy-succinamide ester makes its surface be rich in maleimide base group, then by residual containing multiple cysteines
The construction unit forerunner body protein of base carries out first layer assembling.
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| CN201710637787.6A CN107299107A (en) | 2017-07-31 | 2017-07-31 | A kind of preparation method of protein assembling functional material layer by layer |
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| CN201710637787.6A CN107299107A (en) | 2017-07-31 | 2017-07-31 | A kind of preparation method of protein assembling functional material layer by layer |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113045633A (en) * | 2019-12-27 | 2021-06-29 | 北京大学 | Design of protein heterogeneous entanglement primitive and preparation method of complex catenane structure |
| CN114732898A (en) * | 2022-04-01 | 2022-07-12 | 中国人民解放军军事科学院军事医学研究院 | CpG adjuvant and antigen fixed-point covalent binding method |
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| WO2016193746A1 (en) * | 2015-06-05 | 2016-12-08 | Oxford University Innovation Limited | Methods and products for fusion protein synthesis |
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| WO2016193746A1 (en) * | 2015-06-05 | 2016-12-08 | Oxford University Innovation Limited | Methods and products for fusion protein synthesis |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113045633A (en) * | 2019-12-27 | 2021-06-29 | 北京大学 | Design of protein heterogeneous entanglement primitive and preparation method of complex catenane structure |
| 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 |
| CN114732898A (en) * | 2022-04-01 | 2022-07-12 | 中国人民解放军军事科学院军事医学研究院 | CpG adjuvant and antigen fixed-point covalent binding method |
| CN114732898B (en) * | 2022-04-01 | 2023-05-09 | 中国人民解放军军事科学院军事医学研究院 | Fixed-point covalent binding method of CpG adjuvant and antigen |
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Application publication date: 20171027 |