CN106932375A - The bio-orthogonal Raman in-situ detection method of protein conformation change - Google Patents
The bio-orthogonal Raman in-situ detection method of protein conformation change Download PDFInfo
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- CN106932375A CN106932375A CN201511009815.7A CN201511009815A CN106932375A CN 106932375 A CN106932375 A CN 106932375A CN 201511009815 A CN201511009815 A CN 201511009815A CN 106932375 A CN106932375 A CN 106932375A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
- G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
Abstract
The present invention relates to the bio-orthogonal Raman in-situ detection method of protein conformation change, more particularly to a kind of use bio-orthogonal Raman reporter group specificity mark, the method for detection protein conformation change, it comprises the following steps：A) alpha-non-natural amino acid for carrying bio-orthogonal Raman reporter group is prepared；B) gene expansion technique is utilized, alpha-non-natural amino acid prepared by step a) inserts target protein, so as to realize carrying out target protein bio-orthogonal Raman reporter group mark；C) protein that step b) is obtained is detected using bio-orthogonal Raman technology.The method of the present invention passes through gene codon amplification technique and the enhanced methods of SERS, change that is sensitive, efficiently detecting its conformation under albumen varying environment condition or reactive conditions with bio-orthogonal group, realizes contacting between protein conformation and function in parsing solution and cell system.
Description
Technical field
Biology is being utilized the present invention relates to the detection of protein chemistry and conformation change, more particularly to one kind just
Raman reporter group specificity mark, detection protein are handed over, is examined in vitro using bio-orthogonal Raman technology
Protein conformation change is surveyed, and using bio-orthogonal Raman technology in living cells in situ detection protein conformation
The method of change.
Background technology
The space conformation of protein molecule is its function, the basis of activity.Protein is to environment and stimulation
Response be change by protein or protein complexes its conformations and then realize its function and work
The regulation of property.The position of some of which important amino acid, orientation, motion protein destructurization process
Middle generation significant change.Thus realize that quick, sensitive and efficient detection protein conformation change is (special
It is not the conformation change of in situ detection protein in physiological conditions) for research protein structure and work(
Can contact most important.
At present, the method for research protein structure has many kinds, including X-ray crystallography, nuclear-magnetism
Resonance method, infra-red sepectrometry, fluorescent spectrometry, Raman spectroscopy, circular dichroism detector, mass spectrography etc..
The scope that these methods cut both ways and are applicable.Such as, X-ray crystal diffraction has been developed tens
Year, sample need not be marked, and molecular weight is not limited, Data acquisition and issuance relative maturity, and it can be carried
For the protein structure of atom definition；But the requirement of X-ray crystallography is to analysing protein
Crystal (it is very complicated and difficult to prepare protein monocrystalline), and require that sample size is big, time-consuming, spirit
Sensitivity is poor, is adapted to stable state, is easily obtained the albumen of crystal, for solution in, instantaneous conformation becomes
Change is difficult to be applicable.Infra-red sepectrometry sensitivity is low (to require that content typically should be greater than in component analysis
0.1%), by the signal serious interference of water, the near infrared technology of development also failed to solve the problem later,
So that infra-red sepectrometry is not used to the system close to physiological environment.It is nuclear magnetic resonance method, mass spectrography, glimmering
Light spectroscopic methodology and Raman spectroscopy can detect the protein conformation change in solution at present.But nuclear-magnetism is total to
The method of shaking is appropriate only for sample of the molecular weight less than 40kDa, and sensitivity is low, and analytical concentration generally needs to reach
MM grades, and data processing complex, analysis cost is high.Mass spectrography can grind according to electric charge valence distribution
Study carefully protein conformation, sensitivity is high, can real-time monitoring and quantitative analysis, but conventional ESI-MS with
And the cold spray ionization CSI-MS for improving etc., it is the analysis detection for carrying out gaseous state ion, institute
The information for being given can not sufficiently accurately characterize protein true conformation in the solution.Should in fluorescence method
With it is more be FRET method (FRET), it is necessary to introduce large volume of donor and receive
Body, certain influence can be produced on protein structure and function, and fluorescence method also has stronger utilization present
(effect of such as FRET is comparing sensitive in the range of 10nm, beyond the restrictive condition just very
Hardly possible is applicable).Conversely, Raman spectroscopy is simple and easy to apply, do not disturbed by water, without to detected sample
Product are marked and special preparation, using the teaching of the invention it is possible to provide in the sample such as the molecular composition of material and structure information,
Abundant fingerprint spectral peak is obtained, is applied in cell, tissue even live body.But it is traditional
Detection method based on Raman spectrum, its sensitivity is relatively low, and the peak overlap of bio-molecules,
Serious interference, seriously limits further applying for Raman method.
The content of the invention
The purpose of the present invention is directed to problem present in prior art, there is provided one kind utilizes bio-orthogonal
Raman reporter group specificity mark, detection method of protein.
In order to improve the sensitivity of Raman spectroscopy, Recent study person has developed various enhancing technologies,
Such as surface-enhanced Raman (SERS).SERS is the metal surfaces such as Au, Ag for Raman signal
One kind enhancing phenomenon, can typically realize 106-1012Enhancing effect, can reach and fluorescence spectrum phase
Near sensitivity.On the other hand, overlapped to solve the problems, such as the raman spectra of biomolecule, application
The inventor of people have studied the strategy of orthogonal Raman tag mark, i.e., Raman letter is introduced in biomolecule
Number orthogonal group (such as alkynyl, nitrine, C-D), their Raman signal is in cell Raman signal
Silent region (1800-2800cm-1)。
To reach above-mentioned purpose, use bio-orthogonal Raman reporter group specificity mark of the invention,
Detection method of protein, comprises the following steps：
A) alpha-non-natural amino acid for carrying bio-orthogonal Raman reporter group is prepared；
B) gene expansion technique is utilized, alpha-non-natural amino acid prepared by step a) inserts target protein
Matter, so as to realize carrying out target protein bio-orthogonal Raman reporter group mark；
C) protein that step b) is obtained is detected using bio-orthogonal Raman technology.
Preferably, above-mentioned bio-orthogonal Raman reporter group is alkynyl, nitrine, cyano group or carbon deuterium key.
Preferably, the above method is also using the surface enhanced effect of Gold nanoparticle.
Preferably, passed through using the gold nano grain AuNPs in the surface enhanced effect of Gold nanoparticle
Prepared using reduction of sodium citrate method.
Preferably, HAuCl used in reduction of sodium citrate method4Final concentration of 0.25mM, lemon
Final concentration of 0.01% (w/w) of sour sodium.
Preferably, above-mentioned use bio-orthogonal Raman reporter group specificity is marked, detects protein
Method may also include the affinity for increasing albumen and AuNPs.
It is further preferred that above-mentioned increase albumen includes using containing half Guang ammonia with the affinity of AuNPs
Acid label, the label containing methionine or protein chemistry modification on containing sulfydryl small molecule.
Bio-orthogonal Raman technology vitro detection albumen is used it is a further object of the present invention to provide one kind
The method of conformation change, comprises the following steps：
A) under the conditions of different solutions, using above-mentioned use bio-orthogonal Raman reporter group specificity
Mark, detect method of protein to detect protein conformation change；
B) on the basis of based on step a) detection protein conformation changes, research protein conformation becomes
Change and environment changes relation.
It is in situ in living cells using bio-orthogonal Raman technology it is yet another object of the invention to provide one kind
The method of detection protein conformation change, comprises the following steps：
A) marked using above-mentioned use bio-orthogonal Raman reporter group specificity, detect protein
The protein conformation change of method detection liver cell surface；
B) on the basis of based on step a) detection protein conformation changes, in living cells aspect,
On-spot study protein conformation changes the relation with the stimulated regulation of its protein active.
Preferably, above-mentioned stimulation includes that factors stimulated growth, ligand stimulation or environment change stimulates.
The side of protein is marked, detects in the present invention using bio-orthogonal Raman reporter group specificity
Method, (alkynes is included but is not limited only to using gene expansion technique and carrying bio-orthogonal Raman reporter group
Base, nitrine, cyano group, carbon deuterium key etc.) non-natural amino technic acid realize target protein is carried out
The method of bio-orthogonal Raman reporter group mark.
Using the surface enhanced effect of Gold nanoparticle, realize raw on detection specified protein in the solution
The method of the orthogonal Raman report group of thing.It is specific that the method possesses efficient, sensitive and target protein.
Using bio-orthogonal Raman labels combination Gold nanoparticle surface enhanced effect, realize in living cells
The method that specified protein is detected in aspect.The method good biocompatibility, it is simple to operate, and have concurrently
Efficiently, sensitive and target protein is specific.
Present invention also offers the side changed using bio-orthogonal Raman strategy vitro detection protein conformation
Method；Using the bio-orthogonal Raman reporter group such as alkynyl, with reference to Gold nanoparticle surface enhanced effect,
Under the conditions of different solutions, the method for detection protein conformation change；Based on the inspection of bio-orthogonal Raman
Survey on the basis of protein conformation change, research protein conformation change changes the new side of relation with environment
Method.
Increase the affinity of albumen and AuNPs using the label containing cysteine, increase including other certainly
Plus the method for affinity is used for the enhanced bio-orthogonal Raman strategies detection albumen local conformations of SERS
Change, comprising but be not limited only in the label containing methionine or protein chemistry modification containing small point of sulfydryl
Son.
Present invention also offers use bio-orthogonal Raman strategy in living cells in situ detection protein conformation
The method of change；Using the bio-orthogonal Raman reporter group such as alkynyl, increase with reference to Gold nanoparticle surface
It is potent to answer, the method for detection liver cell surface protein conformation change；Based on the inspection of bio-orthogonal Raman
Survey on the basis of protein conformation change, in living cells aspect, the change of on-spot study protein conformation
The new method of (growth factor, part and environment change etc.) regulation stimulated with its protein active.
The utilization bio-orthogonal Raman reporter group specificity that the present invention is provided is marked, detects protein
Method, the method changed using bio-orthogonal Raman technology vitro detection protein conformation, and using life
The method that the orthogonal Raman technology of thing changes in living cells in situ detection protein conformation, by gene codon
Amplification technique and the enhanced methods of SERS, realize quick, sensitive and efficiently detect with biology just
The change of protein its conformation under different microenvironments or reactive conditions of group is handed over, is particularly realized
The in situ detection of protein conformation change on living cells, and then parse between protein conformation and function
Contact.The technology is based on the method for introducing bio-orthogonal Raman reporter group in protein specific site
Changing very sensitive and with environment change to microenvironment with the molecular vibration signal of Raman group can send out
The theoretical foundation of raw skew, realizes to albumen on the detection of protein conformation change in solution and living cells
The detection in situ and sensitive of matter conformation change.
The above method of the invention is suitable for protein solution and living cells physiological condition lower surface egg
The bio-orthogonal Raman detection of white matter conformation change.The above method of the invention has simple and easy to apply, body
System is close to physiological condition, sensitivity is high, orthogonal Raman signal interference is small, data processing is simple, to egg
White molecular weight is not limited, the characteristics of be adapted to multiple protein system.
Brief description of the drawings
Fig. 1 is the protein conformation change detection based on bio-orthogonal Raman reporter group (alkynyl)；
Fig. 2 is Raman displacement of 35 alkynyls of HdeA albumen under conditions of pH 7；
Fig. 3 is Raman displacement of 35 alkynyls of HdeA albumen under conditions of pH 2；
Fig. 4 is Raman displacement of the HdeA protein 58s position alkynyl under conditions of pH 7；
Fig. 5 is Raman displacement of the HdeA protein 58s position alkynyl under conditions of pH 2；
Fig. 6 (a) is the Raman spectrum that the EGFR (wt-EGFR) being mutated is not on cell；
Fig. 6 (b) is 356 EGFR for being mutated and inserting Penk but stimulate without EGF on cell
Raman spectrum；
Fig. 6 (c) be on cell 356 be mutated and insert the EGFR that Penk stimulates by EGF
Raman spectrum.
Specific embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, below in conjunction with the accompanying drawings
And embodiment, the present invention will be described in further detail.It should be appreciated that described herein specific
Embodiment is only used to explain the present invention, is not intended to limit the present invention.
The specific site insertion on albumen is non-containing orthogonal group using gene amplification for the present invention
Natural amino acid, plasma enhancing can be carried out using Gold nanoparticle to Raman signal, realized to albumen
Efficient, the sensitive and quick detection of matter.Because the Raman signal of group can be by micro near group
The influence of (such as, solution type, radical species, electrical etc.), and there is the skew of Raman peaks position,
Realize the detection to its micro change of the orthogonal reporter group of Raman.And the orthogonal reporter group of Raman is micro-
The change of environment is seen, is associated with the conformation change of protein, this change reflects protein
The transformation of conformation.When after (protein and golden nanometer particle are less than into 10nm), detection is different
Under the conditions of, bio-orthogonal Raman reporter group (being herein alkynyl) Raman signal obtains the target egg
The information of white matter conformation change.It is specifically shown in schematic diagram 1.
The strategy of bio-orthogonal Raman tag mark, i.e., introduce Raman signal orthogonal basis in biomolecule
Group (such as alkynyl, nitrine, C-D), silent region of their Raman signal in cell Raman signal
(1800-2800cm-1).This strategy solves the problems, such as that the raman spectra of biomolecule is overlapped,
Enormously simplify the analysis process of Raman data.Gene codon amplification technique is using in eucaryote
Terminator codon TAG not coded amino acids and using the low property of probability, introduce by screening with
TAG and the orthogonal aminoacyl transferases-tRNA pairs of alpha-non-natural amino acid, make cell by alpha-non-natural amino acid
Insert the TAG mutational sites of albumen.To contain the non-of the orthogonal group of Raman using gene amplification
In natural amino acid insertion albumen, bio-orthogonal Raman detection is carried out.
The detection of conformation change of the albumen of HdeA containing alkynyl under different pH in the solution of embodiment 1
HdeA is the molecular chaperone protein in bacterial membrane interstitial.Under the physiological environment of pH 7, HdeA
Exist in homologous dimerization form；And HdeA depolymerization exposes its active sites in the physiological environment of pH 2
Point is combined with its substrate protein.HdeA is in disorder (confusion) in the physiological environment of pH 2
State, it is impossible to study its structure and conformation change using X-ray crystallography.Penk is to contain
There is the lysine derivative (as shown below) of alkynyl, orthogonal Penk-MbPylRS/tRNA can be used
Penk is inserted the TAG sites of albumen.
The synthesis of AuNPs：Gold nano grain (AuNPs) colloidal sol is prepared using reduction of sodium citrate method,
Wherein HAuCl4Final concentration of 0.25mM, final concentration of 0.01% (w/w) of sodium citrate.
In the ultra-pure water for boiling, it is stirred vigorously down and is separately added into HAuCl4And sodium citrate, after 30min
Stop heating.
Expression and purification containing alkynyl HdeA：Introduced containing many after bacterium HdeA n-end of albumen signal peptides
The label (GCCPGCCGGSGS) of individual cysteine, His6 labels are introduced at C- ends.It 35
Position or 58 amino acids correspondence Codon sequences sport TAG, introduce the non-natural amino containing alkynyl
Sour Penk.In the HdeA gene orders insertion pBAD carriers that will be transformed.Egg is carried out using E.coli
White expression, in the presence of alpha-non-natural amino acid, uses arabinose induced expression.Then use
The albumen of Ni posts purifying expression.Obtain the HdeA albumen containing alkynyl.
AuNPs and HdeA is incubated：HdeAs of the 0.1mg/ml containing alkynyl is added in AuNPs solution,
It is combined using the label and AuNPs of cysteine.After incubation at room temperature 0.5h, 10x is added to delay
Rush solution make the final concentration of 1xPBS of cushioning liquid (pH 7) or sodium citrate buffer (10mM,
NaCl containing 150mM, pH 2).Then the Raman of solution is detected using Horiba Raman microscopes
Spectrum.
Raman detection result is as follows：
1st, albumen HdeA (35 alkynyls), pH 2 to 7
As shown in Fig. 2 under conditions of pH 7, HdeA is in the inactive state of homologous dimerization,
Now the Raman peaks of 35 alkynyls are single peak 2120.4cm-1.As shown in figure 3, and in pH
Under conditions of 2, HdeA depolymerization is monomer, gradually forms activated conformation, now 35 alkynyls
There is the acromion of hangover in Raman peaks, can be with swarming as 2126.1cm-1And 2146.7cm-1.It is all relative
In 2120.4cm-1To wave number displacement high, the structure of " beginning depolymerization " and " nearly activation " is corresponded respectively to
As.
2nd, albumen HdeA (58 alkynyls), pH 2 to 7
As shown in Figures 4 and 5, the conformation change and the potential differences of HdeA 35 of HdeA58 alkynyl are few.
But the two " beginning depolymerization " is different with the ratio of " nearly activation " conformation.Because HdeA is in depolymerization
The change for gradually forming different aminoacids site during activated conformation is not exclusively synchronous, so different
" the beginning depolymerization " of amino acid sites reflection is different with the ratio of " nearly activation " conformation.The present invention is carried
Marked using bio-orthogonal Raman reporter group specificity for one kind, detect method of protein, can
Conformation change of the research different aminoacids site in protein conformation change procedure whether there is, speed and ratio
Example.
The detection of cell surface EGFR containing the alkynyl albumen of embodiment 2 conformation change before and after EGF stimulates
EGFR is epidermal growth factor receptor protein, positioned at surface of cell membrane.EGFR signal paths
The physiology courses such as growth, propagation and differentiation to cell play an important role, close with kinds cancer
It is related.EGFR can occur dimerization after being combined with EGF EGF, main after EGF releases
Exist with monomeric form.
The synthesis of AuNPs：Prepared using reduction of sodium citrate method.
Expression of the EGFR containing alkynyl on cell：By the 210 of EGFR albumen, 250,356 or
The codon of 440 sports TAG respectively.In the presence of alpha-non-natural amino acid Penk, will contain
There are the EGFR and Penk-MbPylRS/tRNA of transformationPyl CUAThe plasmid wink of gene order is transferred to HEK
In 293T cells.After 40h, the Nature enemy of 8h is carried out to cell with the culture medium without serum,
Afterwards with the EGF solution stimulation test group cells of 100ng/ml, 4% paraformaldehyde is used after 15min
Solution fixes cell.10 × PBS of 1/10 volume is added in aurosol, cell system is rapidly joined
Middle incubation 1h.The Raman spectrum of cell is detected using Nanophton Raman microscopes.
Raman results are as follows：
As shown in Fig. 6 (a) -6 (c), EGFR on the cell of (EGF-) is not stimulated with EGF
Main to exist with monomeric form, the cell EGFR for stimulating (EGF+) through EGF is mainly deposited with dimer
In the chemical environment (such as electric field, hydrophilic and hydrophobic) in two kinds of conformations around the alkynyl in same site
Difference, the position of its Raman signal can shift.By taking 356 sites as an example, do not do what is be mutated
Wildtype-EGFR control groups do not have the Raman signal of alkynyl, 356 realities for being mutated and inserting Penk
Testing group can measure the Raman signal of alkynyl, wherein, the alkynyl Raman signal of EGF- group cells exists
2122cm-1, the alkynyl Raman signal of EGF+ group cells is in 2126cm-1, i.e. before and after EGF stimulates
EGFR conformation changes cause the Raman signal of alkynyl to there occurs displacement.4 systems of different loci mutation
Meter result is as shown in following table, wherein 250,356 and 440 sites, Raman signal is high after EGF stimulates
Wave number displacement, and 210 site lower wave number displacements, the chemical environment for reflecting EGFR different locis exist
Different variation tendencies are experienced in Conformation transition.
Table 1 shows Raman position of the different loci alkynyl of EGFR on cell before and after EGF stimulates
Move, it is specific as follows：
Table 1
The result of solution and cell shows, inserts the alkynyl of albumen Raman signal at different conditions
Change can reflect that protein conformation changes.Protein conformation can be changed using bio-orthogonal Raman technology
Carry out in situ detection.
Although the present invention is disclosed as above with preferred embodiment, so it is not limited to the present invention, appoints
What person of ordinary skill in the field, without departing from the spirit and scope of the present invention, when can do some
Perhaps change with improve, therefore protection scope of the present invention depending on as defined in claim when being defined.
Claims (10)
- It is 1. a kind of to be marked using bio-orthogonal Raman reporter group specificity, detect method of protein, It is characterised in that it includes following steps：A) alpha-non-natural amino acid for carrying bio-orthogonal Raman reporter group is prepared；B) gene expansion technique is utilized, alpha-non-natural amino acid prepared by step a) inserts target protein Matter, so as to realize carrying out target protein bio-orthogonal Raman reporter group mark；C) protein that step b) is obtained is detected using bio-orthogonal Raman technology.
- 2. the method for claim 1, it is characterised in that the bio-orthogonal Raman reporter Group is alkynyl, nitrine, cyano group or carbon deuterium key.
- 3. the method for claim 1, it is characterised in that methods described is also using gold The surface enhanced effect of nanoparticle.
- 4. method as claimed in claim 3, it is characterised in that the utilization Gold nanoparticle Gold nano grain AuNPs in surface enhanced effect is prepared by using reduction of sodium citrate method.
- 5. method as claimed in claim 4, it is characterised in that used in reduction of sodium citrate method HAuCl4Final concentration of 0.25mM, final concentration of 0.01% (w/w) of sodium citrate.
- 6. method as claimed in claim 3, it is characterised in that methods described includes increasing albumen With the affinity of AuNPs.
- 7. method as claimed in claim 6, it is characterised in that the increase albumen and AuNPs Affinity include being modified using containing the label of cysteine, the label containing methionine or protein chemistry The upper small molecule containing sulfydryl.
- 8. a kind of method that use bio-orthogonal Raman technology vitro detection protein conformation changes, it is special Levy and be, comprise the following steps：A) under the conditions of different solutions, egg is detected using the method any one of claim 1-7 White matter conformation change；B) on the basis of based on step a) detection protein conformation changes, research protein conformation becomes Change and environment changes relation.
- 9. the side that a kind of use bio-orthogonal Raman technology changes in living cells in situ detection protein conformation Method, it is characterised in that comprise the following steps：A) the albumen texture of liver cell surface is detected using the method any one of claim 1-7 As change；B) on the basis of based on step a) detection protein conformation changes, in living cells aspect, On-spot study protein conformation changes the relation with the stimulated regulation of its protein active.
- 10. method as claimed in claim 9, it is characterised in that the stimulation includes growth factor Stimulate, ligand stimulation or environment change stimulation.
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