CN110292916A - A kind of phosphatide nanometer plate chromatography media and its preparation method and application - Google Patents
A kind of phosphatide nanometer plate chromatography media and its preparation method and application Download PDFInfo
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Abstract
The present invention relates to a kind of phosphatide nanometer plate chromatography medias and its preparation method and application.The phosphatide nanometer plate chromatography media includes charge type phosphatide nanometer plate and affinity substrate；The charge type phosphatide nanometer plate includes phosphatide and the membrane scaffold proteins with affinity tag；The charge type phosphatide nanometer plate is connected by affinity tag with affinity substrate.The phosphatide nanometer plate chromatography media is provided simultaneously with binding ability and protective capability to memebrane protein, and the practical systems containing memebrane protein is made to realize that purifying and one step of reconstruct are completed on chromatographic column；And there is invertibity and regenerability；The problem of which solve protein active losses in traditional memebrane protein investigative technique serious, step redundant and complicated, operation time and effort consuming, easy to operate efficient, memebrane protein activity recovery promotes 20 times or more, and the processing time shortens 95% or more.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to and it is a kind of for isolating and purifying the chromatography media of memebrane protein, especially
It is related to a kind of phosphatide nanometer plate chromatography media and its preparation method and application.
Background technique
The main undertaker of the memebrane protein as biomembrane function, the vital movement of only individual cells does not provide stable interior
Environment has also mediated the connection between cell and cell, cell and matrix, thus transport in the signal transduction of life entity, physiology,
Enzymatic connects etc. with structure plays abundant and important function.Therefore, memebrane protein is always biology, medicine and material
The research emphasis in field.But memebrane protein is also generally acknowledged Research Challenges.Most memebrane proteins are all embedded in biomembrane film layer
Inside, content is very low, and hydrophobicity is extremely strong, isolates and purifies extremely difficult.Conventional method is to use exhibiting high surface activating agent will
Memebrane protein is dissociateed to come from film layer, then is purified through more chromatographic enrichment technology.But a large amount of uses of surfactant, easily lead
Memebrane protein inactivation is caused, physiological structure and function are destroyed serious.
It is the current main policies for restoring Membrane protein conformation and function that memebrane protein, which is reconstructed,.By by film after purification
Albumen reinserts inside adipose membrane, protects the structure and function of memebrane protein, occurs after capable of effectively avoiding memebrane protein from being detached from film layer
Deactivation phenomenom.Memebrane protein reconstructed volume owner will include liposome, the micro- packet of duplicature and nanometer plate.With other two kinds of system phases
It is that membrane scaffold proteins (abbreviation MSP) mix incubation a period of time with the phosphatide being dissolved in surfactant and take off again than, nanometer plate
Except self assembly forms after surfactant, has the characteristics that structure-controllable, surfactant-free interference, has good stability, in film
Albumen research field application effect is brilliant.
This tradition research method for first purifying membrane protein, it being reconstructed again is asked there are following in the prior art
Topic: 1, either which kind of reconstitituted form only has defencive function to memebrane protein, and without binding ability, so that memebrane protein is pure
Changing with reconstruct is the process isolated, time and effort consuming cumbersome tediously long so as to cause whole operation process；2, loss of activity is big, with matter
For memebrane protein, after a series of purification steps, protein active is only 30% or so, and restructuring procedure is at most also only by its activity
It improves to 60%, that is to say, that use this step-by-step processing method reconstructed again after purification, memebrane protein activity is still lost nearly
Half；3, research face is narrow, and the memebrane protein after reconstruct is bonded to stromal surface, can not remove, subsequent to take for this
The a certain particular studies technology of type matrix studies it, and it is few to obtain information content.
Currently, there is no the chromatography system that can be realized memebrane protein purifying and reconstruct one step completion in the prior art, it is based on this,
It is badly in need of developing a kind of new memebrane protein research strategy, to reach memebrane protein efficiently purifying and the active mesh kept while realizing
Mark.
Summary of the invention
In view of the deficiencies of the prior art, the purpose of the present invention is to provide a kind of phosphatide nanometer plate chromatography media and its preparations
Methods and applications, the phosphatide nanometer plate chromatography media are provided simultaneously with binding ability and protective capability to memebrane protein, overcome tradition
Reconstitituted form only protects the deficiency without purifying function to memebrane protein.
In order to achieve that object of the invention, the invention adopts the following technical scheme:
On the one hand, the present invention provides a kind of phosphatide nanometer plate chromatography media, and the phosphatide nanometer plate chromatography media includes electricity
Lotus type phosphatide nanometer plate and affinity substrate；The charge type phosphatide nanometer plate includes phosphatide and the membrane support egg with affinity tag
It is white；The charge type phosphatide nanometer plate is connected by affinity tag with affinity substrate.
The present invention is connected using charge type phosphatide nanometer plate as aglucon with affinity substrate, obtains a kind of for memebrane protein purifying
The phosphatide nanometer plate chromatography media completed with one step of reconstruct, after which fills column, to contain memebrane protein
Practical systems loading, memebrane protein are reconstructed guarantor while passing through electrostatical binding by charge type phosphatide nanometer plate aglucon on chromatographic column
Shield, and memebrane protein-nanometer plate system can be used for further studying after being dissociated.
Preferably, the diameter of the charge type phosphatide nanometer plate be 1-100nm, such as 1nm, 5nm, 10nm, 12nm,
20nm, 25nm, 30nm, 40nm, 50nm, 60nm, 80nm or 100nm etc., preferably 5-50nm, further preferred 10-25nm.
The diameter of the charge type phosphatide nanometer plate is to influence final phosphatide nanometer plate chromatography media obtained to purify effect
One of key factor, value, which is taken as 1-100nm, keeps effect preferable, and 5-50nm makes better effect, and 10-25nm is that effect is best
Range, be unfavorable for the coupling of nanometer plate because of space steric effect if excessive, cause nanometer plate coupling density it is low；If too small
It then can not be in conjunction with memebrane protein, to reduce memebrane protein purification efficiency due to dimensional effect.
Preferably, in terms of every mL affinity substrate, the density of the charge type phosphatide nanometer plate is 0.1-25mg, such as
0.1mg, 0.5mg, 1mg, 2mg, 5mg, 10mg, 12mg, 15mg, 20mg or 25mg etc., preferably 1-10mg.
The density of the charge type phosphatide nanometer plate is also to influence final phosphatide nanometer plate chromatography media purifying function obtained
One of key factor of effect, the effect that value is taken as 0.1-25mg is preferable, and 1-10mg is effect optimum range, if excessive easily
Cause memebrane protein because excessively in conjunction with due to lead to its structure irreversible breaking；Memebrane protein binding ability is caused to decline if too small.
Preferably, Zeta potential of charge type phosphatide nanometer plate under the conditions of 7.4 pH is (- 50~50) eV, example
Such as -50eV, -40eV, -30eV, -25eV, -20eV, -10eV, 0eV, 10eV, 25eV or 50eV, preferably (- 25~25) eV.
Zeta potential of charge type phosphatide nanometer plate under the conditions of 7.4 pH is equally to influence final phosphatide obtained
Nanometer plate chromatography media purifies one of key factor of effect, and it is the better range of effect that value, which is taken as (- 50~50) eV, and (-
25~25) eV keeps effect best, and when Zeta potential is excessively high, memebrane protein can be combined excessively, and when Zeta potential is too low, knot
Resultant force is then insufficient, this all can cause it that can not be bound effectively in nanometer plate.
Preferably, the phosphatide includes phosphatidyl choline (lecithin), dipalmitoylphosphatidylethanolamine (DPPE), two palm fibres
Palmitic acid phosphatidyl choline (DPPC), Distearoyl Phosphatidylcholine (DSPC), phosphatidylserine (PS), glycerophosphatide (PG),
Bromination trimethyl -2,3- dioleoyl oxygroup propyl ammonium (DOTAP) or the oily alkenyloxy group propyl ammonium of chlorination trimethyl -2,3- two
(DOTMA) in any one or at least two combination, described at least two combination such as phosphatidyl choline and two palms
The combination of acyl phosphatidyl-ethanolamine, the combination of dipalmitoylphosphatidylcholine and Distearoyl Phosphatidylcholine, phosphatidyl silk ammonia
Combination, phosphatidyl choline and the combination of bromination trimethyl -2,3- dioleoyl oxygroup propyl ammonium etc. of acid and glycerophosphatide, other
The combination of meaning does not just repeat herein one by one.
The membrane scaffold proteins are that Apoliprotein A-1 albumen is engineered designs obtained amphiphilic polypeptide afterwards, these two
Close polypeptide all has preferable structural flexibility, can form nanometer plate in conjunction with phosphatide, such as include 200 amino acid residues
MSP1。
Preferably, the affinity tag includes histidine (His), maltose-binding protein (MBP), streptococcus (Strep)
In glutathione sulfydryl transferase (GST) any one or at least two combination, described at least two combination is for example
The combination of histidine and maltose-binding protein, the combination of streptococcus and glutathione sulfydryl transferase, maltose-binding protein
With it is streptococcic combination etc., any other combination does not just repeat herein one by one.
Preferably, the affinity substrate includes organic material and/or inorganic material.
Preferably, the affinity substrate includes agarose, glucan, cellulose, konjaku glucomannan, polymethylacrylic acid
In ester, polystyrene, polyacrylamide, silica gel or hydroxyapatite any one or at least two combination, it is described at least
Two kinds of the combination combination of such as agarose and glucan, the combination of cellulose and konjaku glucomannan, polystyrene and poly- third
The combination etc. of acrylamide, any other combination just do not repeat herein one by one.
The affinity substrate is prepared by mechanical mixing method, membrane emulsification or gunite.Each preparation method is both needed to
Specific operating parameter is set, for example, it includes stirring the geometric parameters of tank body and blender that mechanical stirring, which needs the parameter being arranged,
Number, agitator speed etc.；It includes membrane tube aperture, excessively film pressure, excessively film number etc. that membrane emulsification, which needs the parameter being arranged,；Gunite
The parameter for needing to be arranged includes jet size, injection pressure etc..
Preferably, the partial size of the affinity substrate is 1-500 μm, such as 1 μm, 5 μm, 10 μm, 20 μm, 30 μm, 50 μm, 80
μm, 100 μm, 200 μm, 300 μm, 400 μm or 500 μm etc., preferably 5-200 μm, further preferred 10-100 μm.
The partial size of the affinity substrate is equally the pass for influencing final phosphatide nanometer plate chromatography media obtained and purifying effect
One of key factor, the effect that value is taken as 1-500 μm is preferable, and 5-200 μm of effect is more preferable, and 10-100 μm keeps effect best,
If being more than this range, memebrane protein purification efficiency will be substantially reduced.Post separation degree is caused to decline this is because partial size is excessive, partial size mistake
It is small that separating rate is caused to be greatly reduced.
In the present invention, the charge type phosphatide nanometer plate passes through affinity tag and the affinity ligand phase in affinity substrate
Even.
Preferably, the affinity ligand includes metal ion, glutathione, amylose or streptavidin variant
In any one or at least two combination, described at least two combination such as metal ion and glutathione combination,
Combination, glutathione and combination of amylose of amylose and streptavidin variant etc., any other combination
Mode does not just repeat herein one by one.
Preferably, the metal ion includes Ni2+、Cu2+、Co2+、Fe2+Or Zn2+In any one or at least two
Combination, described at least two combination such as Ni2+And Cu2+Combination, Cu2+And Co2+Combination, Fe2+And Zn2+Combination etc.,
Any other combination does not just repeat herein one by one.
Preferably, the metal ion is connected by the chelation between chelating agent with affinity substrate.
Preferably, the chelating agent includes iminodiacetic acid (IDA), tricarboxylic methyl ethylenediamine (TED), three second of nitrilo-
Sour (NTA), carboxymethyl asparagic acid (CM-ASP), tetren (TEPA) or carboxymethyl-α, β-diamino succinic acid
(CM-DASA) in any one or at least two combination, described at least two combination such as iminodiacetic acid and three
The combination of carboxymethyl ethylenediamine, the combination of nitrilotriacetic acid and carboxymethyl asparagic acid, iminodiacetic acid and carboxymethyl-α,
The combination etc. of β-diamino succinic acid, any other combination just do not repeat herein one by one.
On the other hand, the present invention provides a kind of preparation method of phosphatide nanometer plate chromatography media as described above, the system
Preparation Method includes the following steps:
(1) immobilized artificial membrane assembles concentration and the membrane scaffold proteins with affinity tag are subjected to hybrid reaction, obtain charge type phosphorus
Rouge nanometer plate；
(2) charge type phosphatide nanometer plate made from step (1) and affinity substrate are subjected to coupling reaction, obtain the phosphatide
Nanometer plate chromatography media.
In the present invention, step (1) the immobilized artificial membrane assembles concentration the preparation method comprises the following steps: by phospholipid solution be dried with nitrogen system
Embrane method or rotary evaporation film legal system obtain immobilized artificial membrane, then immobilized artificial membrane is dissolved with assembles concentration, obtain the immobilized artificial membrane assembles concentration.
Preferably, the assembles concentration is pH 7.0-7.4 (such as pH 7.0, pH 7.1, pH 7.2, pH 7.3 or pH 7.4
Deng) buffer.
Preferably, the assembles concentration includes phosphate buffer, Na2HPO4Citrate buffer solution, KH2PO4- NaOH buffering
Liquid, barbital sodium-hydrochloride buffer or Tris- buffer.
Preferably, contain sodium chloride in the assembles concentration, concentration 0.02-0.1M, such as 0.02M, 0.03M, 0.04M,
0.05M, 0.06M, 0.07M, 0.08M, 0.09M or 0.1M etc..
Preferably, contain sodium taurocholate in the assembles concentration, concentration 0.01-0.5M, such as 0.01M, 0.03M, 0.04M,
0.05M, 0.06M, 0.07M, 0.1M, 0.2M or 0.5M etc..
In the present invention, when the mixing of step (1) the immobilized artificial membrane assembles concentration and the membrane scaffold proteins with affinity tag
Between be 0.5-12h, such as 0.5h, 1h, 2h, 4h, 5h, 6h, 8h, 10h or 12h etc., preferably 1-8h, further preferred 2-4h.
Preferably, step (1) the immobilized artificial membrane assembles concentration and the mixing temperature of the membrane scaffold proteins with affinity tag are
4-60 DEG C, such as 4 DEG C, 10 DEG C, 20 DEG C, 25 DEG C, 30 DEG C, 40 DEG C, 50 DEG C or 60 DEG C etc., preferably 20-60 DEG C, further preferably
30-50℃。
Preferably, the molar ratio of the membrane scaffold proteins in step (1) the immobilized artificial membrane assembles concentration and phosphatide is 1:(10-
100), such as 1:10,1:20,1:30,1:40,1:50,1:60,1:70,1:80,1:90 or 1:100 etc., preferably 1:(20-60).
Preferably, step (2) coupling refers to is coupled by metal-chelating effect or covalent effect.
Preferably, the time of step (2) described coupling reaction be 10-30h, such as 10h, 12h, 18h, 20h, 22h, for 24 hours,
25h, 26h, 28h or 30h etc..
Preferably, the temperature of step (2) described coupling reaction is 20-40 DEG C, such as 20 DEG C, 22 DEG C, 25 DEG C, 30 DEG C, 32
DEG C, 35 DEG C, 38 DEG C or 40 DEG C etc..
Preferably, before step (2) the progress coupling reaction that charge type phosphatide nanometer plate is saturating in coupling buffer
Analysis extremely balances.
The time that the charge type phosphatide nanometer plate is dialysed in coupling buffer to balance is generally 10-15h, and different
Affinity substrate correspond to different coupling conditions.For example, the coupling condition of metal-chelating effect is usually that charge type phosphatide is received
Rice disk solution is dialysed through the buffer of neutrality/alkalescent (pH 7-8) to balance, wherein it is the most commonly used with phosphate buffer,
And 0.15-1.0M NaCl or other neutral salt can be added, the phosphatide nanometer plate buffering to inhibit non-specific adsorption, after balance
12h is mixed under liquid and metal-chelating medium indoor temp.
About the coupling condition of covalent effect, specifically, for glutathione medium, phosphatide nanometer plate solution is usually
It dialyses in the buffer of neutrality/alkalescent (pH 7-8) to balance, wherein it is the most commonly used with Tris-HCl buffer system, such as 50mM
Tris-HCl, pH 8.0 can add 0.1-0.2M NaCl to inhibit non-specific adsorption；For amylose, phosphatide nanometer
Disk solution is usually dialysed in the buffer of neutrality/alkalescent (pH 7-8) to balance, wherein most with Tris-HCl buffer system
Be it is common, such as 20mM Tris-HCl, 0.2M NaCl, 1mM EDTA, 1mM DTT, pH 7.4；Streptavidin is become
For body, phosphatide nanometer plate solution is usually dialysed in the buffer of neutrality/alkalescent (pH 7-8) to balance, wherein with
Tris-HCl buffer system is the most commonly used, such as 100mM Tris-HCl, 0.15M NaCl, 1mM EDTA, pH 8.0.
As the preferred technical solution of the present invention, the preparation method specifically comprises the following steps:
(1) phospholipid solution then is used immobilized artificial membrane with film method is dried with nitrogen or rotary evaporation film legal system obtains immobilized artificial membrane
Assembles concentration dissolution, obtains the immobilized artificial membrane assembles concentration；
(2) by immobilized artificial membrane assembles concentration made from step (1) with affinity tag membrane scaffold proteins at 4-60 DEG C into
Row hybrid reaction 0.5-12h, the molar ratio of membrane scaffold proteins and phosphatide is 1:(10-100 in immobilized artificial membrane assembles concentration), obtain charge
Type phosphatide nanometer plate；
(3) by charge type phosphatide nanometer plate made from step (2) dialyse in coupling buffer to balance, then with affine base
Matter carries out coupling reaction 18-30h at 20-40 DEG C, obtains the phosphatide nanometer plate chromatography media.
It purifies and reconstructs in memebrane protein in another aspect, the present invention provides a kind of phosphatide nanometer plate chromatography media as described above
In application.
Preferably, the method for the application are as follows: by the phosphatide nanometer plate chromatography media successively through filling column, balance, albumen
Purifying and reconstruct to memebrane protein are realized in loading, elution, elution and cleaning.
The condition of the balance are as follows: after medium fills column, with the equilibration buffer chromatographic column of 5-10 column volume, until flowing
Liquid conductance and pH are constant (consistent with equilibrium liquid) out, and equilibration buffer should be according to the stability and isoelectric point of target protein, ion
The type of exchange media is screened and is optimized.
The condition of the albumen loading are as follows: the buffer of sample is consistent with equilibration buffer, and solid sample equilibrium liquid is molten
Solution is prepared, and low concentration sample solution dialyses or add the salt of corresponding amount with equilibrium liquid, and enriched sample solution is diluted with equilibrium liquid.
The condition of the elution are as follows: eluted with equilibration buffer to baseline.
The condition of the elution are as follows: eluted with elution buffer, will be acted on by metal-chelating and covalent effect is bound to
Memebrane protein-nanometer plate system on affinity substrate disintegrates down, and collects efflux.For receiving by metal-chelating effect preparation
Rice disk medium, is eluted using the buffer containing imidazoles；For the nanometer plate medium prepared by covalent effect, such as paddy
The sweet peptide type of Guang, is eluted, for another example Amylose-type using the buffer containing glutathione, using the buffering containing maltose
Liquid is eluted, for another example streptavidin type, is eluted using the buffer containing desthiobiotin.
The condition of the cleaning are as follows: remaining sediment, albuminate and Fei Te on medium are washed away using cleaning buffer solution
Anisotropic adhesion protein.For example, being cleaned using 70% ethyl alcohol, 30% isopropanol or 0.05-1.0M NaOH solution, then rushed through pure water
It washes.
Compared with the existing technology, the invention has the following advantages:
(1) firstly, phosphatide nanometer plate chromatography media according to the present invention is provided simultaneously with binding ability and guarantor to memebrane protein
Shield ability makes the practical systems containing memebrane protein realize that purifying and one step of reconstruct are completed on chromatographic column, not only maintains tradition
Defencive function possessed by nanometer plate, and nanometer plate have certain quantity of electric charge, can with memebrane protein with electrostatic interaction in conjunction with,
Play the function of purifying protein；
(2) secondly, phosphatide nanometer plate chromatography media according to the present invention is applied to protein techniques field, tradition is solved
Protein active loss is serious in memebrane protein investigative technique, step redundant and complicated, operates the problem of time and effort consuming, height easy to operate
Effect, memebrane protein activity recovery promote 20 times or more, and the processing time shortens 95% or more；
(3) again, phosphatide nanometer plate chromatography media according to the present invention has invertibity and regenerability.Phosphatide nanometer
There is invertibity to take certain elution requirement that will be loaded with after nanometer plate is in conjunction with memebrane protein for the combination of disk and affinity substrate
After the nanometer plate of memebrane protein is disintegrated down from affinity substrate, on the one hand, it is this and meanwhile realize memebrane protein purifying and active protection
The memebrane protein of function-nanometer plate system can be directly used for the structure and function research of memebrane protein, total suitable for surface plasma
A variety of memebrane proteins such as vibration (SPR), nuclear magnetic resonance (NMR), electrochemical impedance spectroscopy (EIS) and atomic force microscope (AFM) study skills
Art；On the other hand, the matrix after dissociating nanometer plate can continue coupled to Nano disk, reuse after regeneration, substantially increase Jie
The service life of matter, application field are wider.
(4) simultaneously, the phosphatide nanometer plate chromatography media according to the present invention advantage controllable with structure and performance.Film egg
White many kinds of, structure is complicated, its activation characteristics of different memebrane proteins differ all very big with purification requirements.By adjusting nanometer disc layer
Medium preparation condition is analysed, realizes that size and distribution, the electrification structures such as property and density and performance parameter of nanometer plate are controllable, thus
Be conducive to the chromatography media type for selecting to facilitate that it is efficiently combined and activity is kept according to memebrane protein practical systems feature.Its
In, the controllable preparation of nanometer plate size and distribution is conducive to it and is combined to different size of memebrane protein, electrification property with it is close
The controllable preparation of degree is conducive to it and is combined to the memebrane protein of different isoelectric points, and the extensive affinity substrate of type is conducive to meet
Different types of chromatography system, thus finally realize nanometer plate chromatography media to different memebrane protein systems it is efficient protection with it is pure
Change.
Detailed description of the invention
Fig. 1 is application signal of the phosphatide nanometer plate chromatography media according to the present invention in memebrane protein is purified and reconstructed
Figure；
Fig. 2 is the particle diameter distribution and Zeta potential figure of nanometer plate in phosphatide nanometer plate chromatography media made from embodiment 1；
Fig. 3 is the transmission electron microscope picture of nanometer plate in phosphatide nanometer plate chromatography media made from embodiment 1；
Fig. 4 is the particle diameter distribution and Zeta potential figure of nanometer plate in phosphatide nanometer plate chromatography media made from embodiment 2；
Fig. 5 is the transmission electron microscope picture of nanometer plate in phosphatide nanometer plate chromatography media made from embodiment 2；
Fig. 6 is the particle diameter distribution and Zeta potential figure of nanometer plate in phosphatide nanometer plate chromatography media made from embodiment 3；
Fig. 7 is the transmission electron microscope picture of nanometer plate in phosphatide nanometer plate chromatography media made from embodiment 3；
Fig. 8 is the chromatography map in embodiment 9；
Fig. 9 is the CO differential spectrum figure in embodiment 9；
Figure 10 is electrophoretic analysis (SDS-PAGE) result figure in embodiment 9；
Figure 11 is the gel filtration chromatography figure in embodiment 17；
Figure 12 is the SDS-PAGE analysis result figure in embodiment 17；
Figure 13 is the Native-PAGE analysis result figure in embodiment 17；
Figure 14 is that (a is cellular membrane disruption liquid, and b is elution after chromatography media for transmission electron microscope picture in embodiment 17
Liquid).
Specific embodiment
The technical scheme of the invention is further explained by means of specific implementation.Those skilled in the art should be bright
, the described embodiments are merely helpful in understanding the present invention, should not be regarded as a specific limitation of the invention.
Detection involved in following embodiments include the following:
(1) nanometer plate partial size and distribution and Zeta potential measure: being measured using dynamic light scattering.
(2) morphology observation nanometer plate morphology analysis: is carried out to the nanometer plate that classification is collected using transmission electron microscope.
(3) front and back nanometer plate density measurement: is coupled using Coomassie Brilliant Blue measurement nanometer plate solution and affinity substrate
Supernatant protein concentration calculates nanometer plate density according to the following formula.
Nanometer plate density=(C1V1-C0V0)/V
In formula, C0、C1It is clearance response front and back protein concentration (mg/mL) on coupling liquid, V respectively0、V1It is on coupling liquid respectively
Volume (mL) before and after clearance response, V is affinity substrate volume (mL).
(4) it the average grain diameter measurement of affinity substrate: is measured using laser particle size analyzer.
(5) CO differential spectrum is tested: sodium dithionite is added in the component collected to gel filtration chromatography, stands
5s.After stable system, baseline scan is carried out from 400nm to 500nm using microplate reader, carbon monoxide is next passed through into system
60s (bubble is not spilt over continuous and solution to be advisable) continues to be scanned using microplate reader from 400nm to 500nm.Experiment is with liver
Microsome is crushed liquid and makees blank control.CYP450 content is calculated according to the following formula:
In formula, △ OD is the OD value difference at wavelength 450nm and 490nm, and 91 be extinction coefficient (mmol/L).
(6) electrophoretic analysis (SDS-PAGE): according to preparative separation glue, concentration glue, sample loading, electrophoresis, dyeing and decoloration
And etc. carry out SDS-PAGE experiment.Resolving gel concentration is 13.5%.Purity quantitative analysis is carried out by scanning densitometer.
Membrane scaffold proteins amino acid sequence involved in following embodiments are as follows: GHHHHHHIEGRLKLLDNWDSVTSTF
SKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQK
LHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKP
ALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ。
Embodiment 1
The present embodiment prepares a kind of phosphatide nanometer plate chromatography media, is named as nanometer plate-Ni-IDA agarose chromatography medium,
Preparation method is as follows:
(1) preparation is connected with the membrane scaffold proteins (abbreviation MSP albumen) of His label: will have the MSP albumen sequence of His label
Column gene monomer is connect with pET-21a, is obtained expression vector, and convert to E.coli BL21, is obtained corresponding genetic engineering
Bacterium.Strain is connected on LB solid medium by plate streak, picking single colonie is gone in LB liquid medium, and 30 DEG C are shaken
Bed is incubated overnight, and is obtained first order seed and is continued to cultivate, obtains secondary seed.When secondary seed OD600 is between 0.8-1.0 into
Bacteria suspension, 4 DEG C of centrifugations are collected in row IPTG induction.Supernatant is chromatographed through Ni-IDA column, loading condition (20mM PB, 0.15M
NaCl, 20mM imidazoles, pH7.4), elution requirement (20mM PB, 0.15M NaCl, 200mM imidazoles, pH7.4) collects eluting peak,
Concentration, 4 DEG C of preservations, obtains the MSP albumen for being connected with His label.
(2) charge type phosphatide nanometer plate is prepared: it is accurate to measure 1mL100mM lecithin soln, it is added in centrifuge tube, through nitrogen
Air-blowing is dry.5mL assembles concentration (20mM phosphate, 0.15M sodium chloride, gallbladder containing 100mM in pH 7.4 are added into obtained immobilized artificial membrane
Sour sodium), 50 DEG C of water-baths.After immobilized artificial membrane is dissolved completely in assembles concentration, the His label membrane support egg of 15mg/mL is added thereto
White (molar ratio of MSP albumen and phosphatide is 1:60), mixes 2h.System is obtained in 4 DEG C of dialysed overnights.
Zeta potential value under the average diameter of nanometer plate obtained, pH 7.4 is measured, as a result as shown in Figure 2:
Nanometer disk diameter is 10nm, and the Zeta potential value under pH 7.4 is -8eV；Transmission electron microscope analysis, knot are carried out to nanometer plate obtained
Fruit is as shown in Figure 3: nanometer disk shape is approximate circle, and regular, no clustering phenomena of arranging.
(3) it prepares phosphatide nanometer plate chromatography media: weighing clean drain of 5g and be situated between by the agarose of affinity ligand of Ni-IDA
Matter is placed in triangular flask, the above-mentioned nanometer plate solution of 5mL is added, at room temperature shaken overnight.After reaction, with a large amount of deionized waters
Nanometer plate-Ni-IDA agarose chromatography medium is made in cleansing medium.
Nanometer plate obtained coupling density is measured as 12mg/mL, the average grain diameter of affinity media is measured as 90 μm.
Embodiment 2
The present embodiment prepares a kind of phosphatide nanometer plate chromatography media, is named as nanometer plate-glutathione-dextran chromatography and is situated between
Matter, preparation method are as follows:
(1) preparation is connected with the membrane scaffold proteins (abbreviation MSP albumen) of GST label: method is pure referring to method in embodiment 1
It is chromatographed when change using glutathione column.
(2) prepare charge type phosphatide nanometer plate: it is accurate measure 1mL100mM lecithin and DPPC combination solution (by mole
Compounded than 1:1), it is added in centrifuge tube, through being dried with nitrogen.Into obtained immobilized artificial membrane be added 5mL assembles concentration (20mM phosphate,
0.15M sodium chloride, sodium taurocholate containing 100mM in pH 7.4), 50 DEG C of water-baths.After immobilized artificial membrane is dissolved completely in assembles concentration, thereto
The GST label membrane scaffold proteins (molar ratio of MSP albumen and phosphatide is 1:10) of 1mg/mL are added, mix 2h.System is obtained in 4
DEG C dialysed overnight.
Zeta potential value under the average diameter of nanometer plate obtained, pH 7.4 is measured, as a result as shown in Figure 4:
Nanometer disk diameter is 1nm, and the Zeta potential value under pH 7.4 is -1eV；Transmission electron microscope analysis, knot are carried out to nanometer plate obtained
Fruit is as shown in Figure 5: nanometer disk shape is approximate circle, and regular, no clustering phenomena of arranging.
(3) it prepares phosphatide nanometer plate chromatography media: weighing 5g and clean the glucan drained using glutathione as affinity ligand
Medium is placed in triangular flask, the above-mentioned nanometer plate solution of 5mL is added, at room temperature shaken overnight.After reaction, with a large amount of deionizations
Nanometer plate-glutathione-dextran chromatography medium is made in water cleansing medium.
Nanometer plate obtained coupling density is measured as 0.1mg/mL, the average grain diameter of affinity media is measured as 60 μ
m。
Embodiment 3
The present embodiment prepares a kind of phosphatide nanometer plate chromatography media, is named as nanometer plate-amylose-konjaku glucomannan
Chromatography media, preparation method are as follows:
(1) preparation is connected with the membrane scaffold proteins (abbreviation MSP albumen) of maltose-binding protein (MBP) label: method reference
Method in embodiment 1 is chromatographed using amylopectin column when purifying.
(2) charge type phosphatide nanometer plate is prepared: the accurate lecithin and phosphatidyl silk for measuring 1mL integral molar quantity and being 100mM
The combination solution (1:5 is compounded in molar ratio) of propylhomoserin (PS), is added in centrifuge tube, through being dried with nitrogen.Into obtained immobilized artificial membrane
5mL assembles concentration (20mM phosphate, 0.15M sodium chloride, sodium taurocholate containing 100mM in pH 7.4), 50 DEG C of water-baths are added.To immobilized artificial membrane
After being dissolved completely in assembles concentration, the MBP label membrane scaffold proteins (molar ratio of MSP albumen and phosphatide of 30mg/mL is added thereto
For 1:100), 2h is mixed.System is obtained in 4 DEG C of dialysed overnights.
Zeta potential value under the average diameter of nanometer plate obtained, pH 7.4 is measured, as a result as shown in Figure 6:
Nanometer disk diameter is 100nm, and the Zeta potential value under pH 7.4 is -50eV；Transmission electron microscope analysis is carried out to nanometer plate obtained,
As a result as shown in Figure 7: nanometer disk shape is approximate circle, and regular, no clustering phenomena of arranging.
(3) it prepares phosphatide nanometer plate chromatography media: weighing 5g and clean the konjaku Portugal drained using amylopectin as affinity ligand
Sweet glycan medium is placed in triangular flask, the above-mentioned nanometer plate solution of 5mL is added, at room temperature shaken overnight.After reaction, with a large amount of
Nanometer plate-amylose-konjaku glucomannan chromatography media is made in deionized water cleansing medium.
Nanometer plate obtained coupling density is measured as 25mg/mL, the average grain diameter of affinity media is measured as 120 μ
m。
Embodiment 4
The present embodiment prepares a kind of phosphatide nanometer plate chromatography media, is named as nanometer plate-streptavidin variant
(Strep-Tactin)-polymethacrylates chromatography media, preparation method are as follows:
(1) preparation is connected with the membrane scaffold proteins (abbreviation MSP albumen) of Strep label: method in method reference embodiment 1,
It is chromatographed when purifying using streptavidin variant column.
(2) prepare charge type phosphatide nanometer plate: the accurate 1mL integral molar quantity that measures is the lecithin of 100mM and answering for DOTAP
With solution (1:10 is compounded in molar ratio), it is added in centrifuge tube, through being dried with nitrogen.5mL assembling is added into obtained immobilized artificial membrane
Liquid (20mM phosphate, 0.15M sodium chloride, sodium taurocholate containing 100mM in pH 7.4), 50 DEG C of water-baths.It is dissolved completely in immobilized artificial membrane
After assembles concentration, the MBP label membrane scaffold proteins (molar ratio of MSP albumen and phosphatide is 1:70) of 12mg/mL are added thereto, mix
Close 2h.System is obtained in 4 DEG C of dialysed overnights.
Zeta potential value under the average diameter of nanometer plate obtained, pH 7.4 is measured, nanometer disk diameter is
Zeta potential value under 12nm, pH 7.4 is 50eV；Carry out transmission electron microscope analysis to nanometer plate obtained: nanometer disk shape is approximate
Circle, and regular, no clustering phenomena of arranging.
(3) prepare phosphatide nanometer plate chromatography media: it is affine for weighing clean drain with streptavidin variant of 5g
The polymethacrylates medium of aglucon is placed in triangular flask, the above-mentioned nanometer plate solution of 5mL is added, at room temperature shaken overnight.Instead
After answering, with a large amount of deionized water cleansing mediums, nanometer plate-streptavidin variant (Strep- is made
Tactin)-polymethacrylates chromatography media.
Nanometer plate obtained coupling density is measured as 10mg/mL, the average grain diameter of affinity media is measured as 50 μm.
Embodiment 5
The present embodiment prepares a kind of phosphatide nanometer plate chromatography media, is named as nanometer plate Cu-CM-ASP silica gel column chromatography medium,
Preparation method is as follows:
(1) preparation is connected with the membrane scaffold proteins (abbreviation MSP albumen) of His label: method is referring to method in embodiment 1.
(2) prepare charge type phosphatide nanometer plate: method is referring to method in embodiment 1.
Zeta potential value under the average diameter of nanometer plate obtained, pH 7.4 is measured, nanometer disk diameter is
Zeta potential value under 10nm, pH 7.4 is -8eV；Carry out transmission electron microscope analysis to nanometer plate obtained: nanometer disk shape is approximate
Circle, and regular, no clustering phenomena of arranging.
(3) it prepares phosphatide nanometer plate chromatography media: weighing clean drain of 5g and be situated between by the silica gel of affinity ligand of Cu-CM-Asp
Matter is placed in triangular flask, the above-mentioned nanometer plate solution of 5mL is added, at room temperature shaken overnight.After reaction, with a large amount of deionized waters
Nanometer plate Cu-CM-ASP silica gel column chromatography medium is made in cleansing medium.
Nanometer plate obtained coupling density is measured as 15mg/mL, the average grain diameter of affinity media is measured as 5 μm.
Embodiment 6
The present embodiment prepares a kind of phosphatide nanometer plate chromatography media, is named as nanometer plate-Ni-IDA agarose chromatography medium-
2, preparation method and the difference of embodiment 1 are only that: the molar ratio of MSP albumen and phosphatide is 1:100.
Zeta potential value under the average diameter of nanometer plate obtained, pH 7.4 is measured, nanometer disk diameter is
Zeta potential value under 100nm, pH 7.4 is -10eV；Carry out transmission electron microscope analysis to nanometer plate obtained: nanometer disk shape is close
Like circle, and regular, no clustering phenomena of arranging.
Embodiment 7
The present embodiment prepares a kind of phosphatide nanometer plate chromatography media, is named as nanometer plate-Ni-IDA agarose chromatography medium-
3, preparation method and the difference of embodiment 1 are only that: accurate to measure the lecithin and phosphatidyl that 1mL integral molar quantity is 100mM
The combination solution of serine (PS) (1:4 is compounded in molar ratio).
Zeta potential value under the average diameter of nanometer plate obtained, pH 7.4 is measured, nanometer disk diameter is
Zeta potential value under 15nm, pH 7.4 is -30eV；Carry out transmission electron microscope analysis to nanometer plate obtained: nanometer disk shape is close
Like circle, and regular, no clustering phenomena of arranging.
Embodiment 8
The present embodiment prepares a kind of phosphatide nanometer plate chromatography media, is named as nanometer plate-Ni-IDA agarose chromatography medium-
4, preparation method and the difference of embodiment 1 are only that: the average grain diameter of affinity media is 200 μm, other are remained unchanged.
Embodiment 9
The present embodiment is used for protein liver particle using nanometer plate-Ni-IDA agarose chromatography medium made from embodiment 1
The purifying of body CYP450, operating method are as follows:
Nanometer plate-Ni-IDA agarose chromatography medium made from embodiment 1 is filled into column (column volume 0.5mL), and is connected to
PPS protein chromatography system.After Buffer A (20mM PB, pH 7.4) balance columns, liquid loading is crushed using hepatomicrosome, is flowed
Fast 0.2mL/min, continuation is eluted with Buffer A, finally with Buffer B (20mM PB, imidazoles containing 200mM, pH 7.4)
It is eluted.
Record chromatographs map, as a result (P as shown in Figure 81And P1' it is respectively that coupled to Nano disc layer analysis media group and not being coupled is received
Rice disc layer analysis media group penetrates peak, P2The eluting peak of media group is analysed for coupled to Nano disc layer), as seen from the figure: non-coupled to Nano
It is eluted through imidazoles without obvious eluting peak after the chromatography media loading of disk, illustrates that it is not bound with albumen, and be coupled nanometer plate
Chromatography media generates apparent eluting peak after imidazole solution elutes.
The eluting peak is collected, is analyzed it using CO differential spectrum, as shown in figure 9, as seen from the figure: nanometer plate chromatography
The eluting peak P of medium2There is obvious absorption at 450nm, and coupled to Nano disc layer analysis media group and the analysis of non-coupled to Nano disc layer are situated between
Matter group penetrates peak P1And P1' all do not absorbed at this.
(1 is crushed liquid supernatant for hepatomicrosome to electrophoretic analysis result, and 2 be membrane scaffold proteins liquid, and 3 is micro- for liver as shown in Figure 10
Plastochondria is crushed liquid rear solution in conjunction with nanometer plate chromatography media, and 4 be the eluting peak of nanometer plate chromatography media, and 5 be non-coupled to Nano
Disk chromatography media penetrates peak): nanometer plate medium eluting peak has obvious band near molecular weight 50,000, illustrates using the medium
It can achieve the effect that purify CYP450.Eluting peak CYP450 content is 0.04nmol/mg, electrophoresis purity > 90%.The result shows that
The medium plays the role of activity holding while capable of purifying to CYP450.
Embodiment 10
The present embodiment is micro- for protein liver using nanometer plate-glutathione-dextran chromatography medium made from embodiment 2
The purifying of plastochondria CYP450, operating method are as follows:
Nanometer plate-glutathione prepared by embodiment 2-dextran chromatography medium fills column, and step is the same as embodiment 9.Elution
When in sample-loading buffer be added 20mM reduced glutathione, collect eluting peak.CO differential spectrum and electrophoresis point are carried out respectively
Analysis.After measured, eluting peak CYP450 content is 0.02nmol/mg, electrophoresis purity > 90%.The result shows that the medium can be right
Play the role of activity holding while CYP450 is purified.
Embodiment 11
The present embodiment is used for albumen using nanometer plate-amylose-konjaku glucomannan chromatography media made from embodiment 3
The purifying of matter liver cytochrome 450, operating method are as follows:
Nanometer plate-amylose prepared by embodiment 3-konjaku glucomannan chromatography media fills column, the same embodiment of step
9.10mM maltose is added when elution in sample-loading buffer, collects eluting peak.CO differential spectrum and electrophoretic analysis are carried out respectively.
After measured, eluting peak CYP450 content is 0.03nmol/mg, electrophoresis purity > 90%.The result shows that the medium can be right
Play the role of activity holding while CYP450 is purified.
Embodiment 12
The present embodiment is poly- using nanometer plate-streptavidin variant (Strep-Tactin)-made from embodiment 4
Layer of methacrylate analyses the purifying that medium is used for protein liver cytochrome 450, and operating method is as follows:
Nanometer plate prepared by embodiment 4-streptavidin variant (Strep-Tactin)-polymethylacrylic acid
Ester chromatography media fills column, and step is with embodiment 9, and wherein pH of buffer is adjusted to (the 25mM NaHCO of pH 11.03).When elution
2.5mM desthiobiotin is added in sample-loading buffer, collects eluting peak.CO differential spectrum and electrophoretic analysis are carried out respectively.Through surveying
Fixed, eluting peak CYP450 content is 0.03nmol/mg, electrophoresis purity > 90%.The result shows that the medium can to CYP450 into
Play the role of activity holding while row purifying.
Embodiment 13
The present embodiment is used for protein liver particle using nanometer plate Cu-CM-ASP silica gel column chromatography medium made from embodiment 5
The purifying of body CYP450, operating method are as follows:
Nanometer plate Cu-CM-ASP silica gel column chromatography medium prepared by embodiment 5 fills column, and step is the same as embodiment 9.When elution
200mM imidazoles is added in sample-loading buffer, collects eluting peak.CO differential spectrum and electrophoretic analysis are carried out respectively.After measured, it elutes
Peak CYP450 content is 0.04nmol/mg, electrophoresis purity > 90%.The result shows that the medium can purify CYP450
Play the role of activity holding simultaneously.
Embodiment 14
The present embodiment is micro- for protein liver using nanometer plate-Ni-IDA agarose chromatography medium -2 made from embodiment 6
The purifying of plastochondria CYP450, operating method are as follows:
Nanometer plate-Ni-IDA agarose chromatography medium prepared by embodiment 6 fills column, and step is the same as embodiment 9.Collect elution
Peak carries out CO differential spectrum and electrophoretic analysis respectively.After measured, eluting peak CYP450 content is 0.02nmol/mg, electrophoresis purity
> 90%.
Embodiment 15
The present embodiment is micro- for protein liver using nanometer plate-Ni-IDA agarose chromatography medium -3 made from embodiment 7
The purifying of plastochondria CYP450, operating method are as follows:
Nanometer plate-Ni-IDA agarose chromatography medium prepared by embodiment 7 fills column, and step is the same as embodiment 9.Collect elution
Peak carries out CO differential spectrum and electrophoretic analysis respectively.After measured, eluting peak CYP450 content is 0.02nmol/mg, electrophoresis purity
> 80%.
Embodiment 16
The present embodiment is micro- for protein liver using nanometer plate-Ni-IDA agarose chromatography medium -4 made from embodiment 8
The purifying of plastochondria CYP450, operating method are as follows:
Nanometer plate-Ni-IDA agarose chromatography medium prepared by embodiment 8 fills column, and step is the same as embodiment 9.Collect elution
Peak carries out CO differential spectrum and electrophoretic analysis respectively.After measured, eluting peak CYP450 content is 0.02nmol/mg, electrophoresis purity
> 90%.
Comparative example 1
This comparative example handles liver cytochrome 450, operating method using conventional method are as follows:
According to first purifying, afterwards the conventional method reconstructed handles memebrane protein, the memebrane protein that will be purified from hepatomicrosome
CYP450 is mixed with amphoteric surfactant CHAPS (3- [3- (gallbladder amido propyl) dimethylamino] propane sulfonic acid inner salt) according to 1:3,
It is stirred overnight at 0 DEG C.Centrifuging and taking supernatant.Molecular sieve prepacked column Superdex200 (10mm I.D.*300mm) is connected into PPS
Protein chromatography system, with Buffer A (20mM PB, 0.03%Chaps, pH 6) balance.After column equilibration, loading, flow velocity
0.2mL/min.Collect each peak.Nanometer plate is added in peak to collecting, is incubated for 0.5h.Incubating Solution is divided using CO differential spectrum
Analysis.The index of embodiment 9-16 and comparative example 1 after purification is summarized into such as table 1, seen from table 1: receiving using phosphatide of the present invention
Rice disc layer analysis medium purification CYP450 either protein content, purity or the process limited is all far superior to comparative example 1.
Table 1
Embodiment 17
The present embodiment is used for cell membrane protein using nanometer plate-Ni-IDA agarose chromatography medium made from embodiment 1
Purifying, operating method is as follows:
Firstly, cellular membrane disruption liquid is carried out analysed by gel filtration chromatography, as shown in figure 11: chromatogram retention volume is certainly
7.0-24.0mL range is crushed liquid and many peaks occurs, these peaks respectively correspond various sizes of membrane component, and ingredient is very multiple
It is miscellaneous, illustrate that the cell membrane protein is difficult to separate using conventional chromatographic methods.
Nanometer plate-Ni-IDA agarose chromatography medium made from embodiment 1 is filled into column (column volume 0.5mL), and is connected to
PPS protein chromatography system.After Buffer A (20mM PB, pH 7.4) balance columns, it is crushed on liquid using Mouse Somatic Cells film
Sample, flow velocity 0.2mL/min, continuation is eluted with Buffer A, finally with Buffer B (20mM PB, imidazoles containing 200mM, pH
7.4) it is eluted, respectively by two kinds of electrophoresis method, that is, SDS-PAGE and Native-PAGE and transmission electron microscope (TEM) to cell
Membrane protein extraction situation is analyzed.
SDS-PAGE analyzes result, and (1 is nanometer plate made from embodiment 1, and 2 be cellular membrane disruption liquid, and 3 are as shown in figure 12
Eluting peak), as seen from the figure: cellular membrane disruption liquid contains multi-ribbon, and component is extremely complex, after the processing of nanometer plate chromatography media,
Eluting peak is made of nanometer plate and memebrane protein, illustrates that epicyte protein is effectively purified.
(1 is membrane scaffold proteins to Native-PAGE analysis result, and 2,3,4,7 be to receive made from embodiment 1 as shown in figure 13
Rice disk, 5 be cellular membrane disruption liquid, and 6 be eluting peak), as seen from the figure: cellular membrane disruption liquid contains multi-ribbon, and component is extremely complex,
After the processing of nanometer plate chromatography media, eluting peak is made of nanometer plate and memebrane protein, illustrates that epicyte protein is effectively purified.
TEM result is (a is cellular membrane disruption liquid, and b is the eluent after chromatography media) as shown in figure 14, as seen from the figure:
Contain a large amount of irregularities in cellular membrane disruption liquid, thus it is speculated that be various complex components on cell membrane, at nanometer plate chromatography media
After reason, ingredient is single, and epicyte protein is incorporated in nanometer plate, realizes purifying.
The Applicant declares that the present invention is explained by the above embodiments phosphatide nanometer plate chromatography media and its system of the invention
Preparation Method and application, but the present invention is not limited to the above embodiments, that is, does not mean that the present invention must rely on above-described embodiment
It could implement.It should be clear to those skilled in the art, any improvement in the present invention, to each raw material of product of the present invention
Equivalence replacement and addition, the selection of concrete mode of auxiliary element etc., all fall within protection scope of the present invention and the open scope
Within.
The preferred embodiment of the present invention has been described above in detail, still, during present invention is not limited to the embodiments described above
Detail within the scope of the technical concept of the present invention can be with various simple variants of the technical solution of the present invention are made, this
A little simple variants all belong to the scope of protection of the present invention.
It is further to note that specific technical features described in the above specific embodiments, in not lance
In the case where shield, can be combined in any appropriate way, in order to avoid unnecessary repetition, the present invention to it is various can
No further explanation will be given for the combination of energy.
SEQUENCE LISTING
<110>Chinese Academy Of Sciences Process Engineering Research Institute
<120>a kind of phosphatide nanometer plate chromatography media and its preparation method and application
<130> 2019
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 211
<212> PRT
<213>artificial synthesized sequence
<400> 1
Gly His His His His His His Ile Glu Gly Arg Leu Lys Leu Leu Asp
1 5 10 15
Asn Trp Asp Ser Val Thr Ser Thr Phe Ser Lys Leu Arg Glu Gln Leu
20 25 30
Gly Pro Val Thr Gln Glu Phe Trp Asp Asn Leu Glu Lys Glu Thr Glu
35 40 45
Gly Leu Arg Gln Glu Met Ser Lys Asp Leu Glu Glu Val Lys Ala Lys
50 55 60
Val Gln Pro Tyr Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu Met
65 70 75 80
Glu Leu Tyr Arg Gln Lys Val Glu Pro Leu Arg Ala Glu Leu Gln Glu
85 90 95
Gly Ala Arg Gln Lys Leu His Glu Leu Gln Glu Lys Leu Ser Pro Leu
100 105 110
Gly Glu Glu Met Arg Asp Arg Ala Arg Ala His Val Asp Ala Leu Arg
115 120 125
Thr His Leu Ala Pro Tyr Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala
130 135 140
Arg Leu Glu Ala Leu Lys Glu Asn Gly Gly Ala Arg Leu Ala Glu Tyr
145 150 155 160
His Ala Lys Ala Thr Glu His Leu Ser Thr Leu Ser Glu Lys Ala Lys
165 170 175
Pro Ala Leu Glu Asp Leu Arg Gln Gly Leu Leu Pro Val Leu Glu Ser
180 185 190
Phe Lys Val Ser Phe Leu Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu
195 200 205
Asn Thr Gln
210
Claims (10)
1. a kind of phosphatide nanometer plate chromatography media, which is characterized in that the phosphatide nanometer plate chromatography media includes charge type phosphatide
Nanometer plate and affinity substrate；The charge type phosphatide nanometer plate includes phosphatide and the membrane scaffold proteins with affinity tag；It is described
Charge type phosphatide nanometer plate is connected by affinity tag with affinity substrate.
2. phosphatide nanometer plate chromatography media as described in claim 1, which is characterized in that the charge type phosphatide nanometer plate it is straight
Diameter is 1-100nm, preferably 5-50nm, further preferred 10-25nm；
Preferably, in terms of every mL affinity substrate, the density of the charge type phosphatide nanometer plate is 0.1-25mg, preferably 1-10mg；
Preferably, Zeta potential of charge type phosphatide nanometer plate under the conditions of pH7.4 be (- 50~50) eV, preferably (- 25
~25) eV.
3. phosphatide nanometer plate chromatography media as claimed in claim 1 or 2, which is characterized in that the phosphatide includes phosphatidyl gallbladder
It is alkali, dipalmitoylphosphatidylethanolamine, dipalmitoylphosphatidylcholine, Distearoyl Phosphatidylcholine, phosphatidylserine, sweet
Appointing in oily phosphatide, bromination trimethyl -2,3- dioleoyl oxygroup propyl ammonium or the oily alkenyloxy group propyl ammonium of chlorination trimethyl -2,3- two
It anticipates a kind of or at least two combinations；
Preferably, the affinity tag includes in histidine, maltose-binding protein, streptococcus or glutathione sulfydryl transferase
Any one or at least two combination.
4. phosphatide nanometer plate chromatography media as claimed in any one of claims 1-3, which is characterized in that the affinity substrate packet
Include organic material and/or inorganic material；
Preferably, the affinity substrate include agarose, glucan, cellulose, konjaku glucomannan, polymethacrylates,
In polystyrene, polyacrylamide, silica gel or hydroxyapatite any one or at least two combination；
Preferably, the partial size of the affinity substrate is 1-500 μm, preferably 5-200 μm, further preferred 10-100 μm.
5. such as phosphatide nanometer plate chromatography media of any of claims 1-4, which is characterized in that the charge type phosphatide
Nanometer plate is connected by affinity tag with the affinity ligand in affinity substrate；
Preferably, the affinity ligand includes in metal ion, glutathione, amylose or streptavidin variant
Any one or at least two combination；
Preferably, the metal ion includes Ni2+、Cu2+、Co2+、Fe2+Or Zn2+In any one or at least two combination；
Preferably, the metal ion is connected by the chelation between chelating agent with affinity substrate；
Preferably, the chelating agent includes iminodiacetic acid, tricarboxylic methyl ethylenediamine, nitrilotriacetic acid, carboxymethyl asparagus fern
Propylhomoserin, tetren or carboxymethyl-α, in β-diamino succinic acid any one or at least two combination.
6. the preparation method of phosphatide nanometer plate chromatography media according to any one of claims 1 to 5, which is characterized in that described
Preparation method includes the following steps:
(1) immobilized artificial membrane assembles concentration and the membrane scaffold proteins with affinity tag are subjected to hybrid reaction, obtain charge type phosphatide and receives
Rice disk；
(2) charge type phosphatide nanometer plate made from step (1) and affinity substrate are subjected to coupling reaction, obtain the phosphatide nanometer
Disk chromatography media.
7. the preparation method of phosphatide nanometer plate chromatography media as claimed in claim 6, which is characterized in that step (1) described phosphorus
Adipose membrane assembles concentration the preparation method comprises the following steps: by phospholipid solution be dried with nitrogen film method or rotary evaporation film legal system obtain immobilized artificial membrane,
Immobilized artificial membrane is dissolved with assembles concentration again, obtains the immobilized artificial membrane assembles concentration；
Preferably, the assembles concentration is the buffer of pH7.0-7.4；
Preferably, the assembles concentration includes phosphate buffer, Na2HPO4Citrate buffer solution, KH2PO4- NaOH buffer, bar ratio
Appropriate sodium-hydrochloride buffer or Tris- buffer；
Preferably, sodium chloride, concentration 0.02-0.1M are contained in the assembles concentration；
Preferably, sodium taurocholate, concentration 0.01-0.5M are contained in the assembles concentration.
8. the preparation method of phosphatide nanometer plate chromatography media as claimed in claims 6 or 7, which is characterized in that step (1) is described
Immobilized artificial membrane assembles concentration with affinity tag membrane scaffold proteins incorporation time be 0.5-12h, preferably 1-8h, further preferably
2-4h；
Preferably, the mixing temperature of step (1) the immobilized artificial membrane assembles concentration and the membrane scaffold proteins with affinity tag is 4-60
DEG C, preferably 20-60 DEG C, further preferred 30-50 DEG C；
Preferably, the molar ratio of the membrane scaffold proteins in step (1) the immobilized artificial membrane assembles concentration and phosphatide is 1:(10-100), it is excellent
Select 1:(20-60)；
Preferably, step (2) coupling refers to is coupled by metal-chelating effect or covalent effect；
Preferably, the time of step (2) described coupling reaction is 10-30h；
Preferably, the temperature of step (2) described coupling reaction is 20-40 DEG C；
Preferably, before step (2) the progress coupling reaction by charge type phosphatide nanometer plate dialyse in coupling buffer to
Balance.
9. the preparation method of the phosphatide nanometer plate chromatography media as described in any one of claim 6-8, which is characterized in that described
Preparation method specifically comprises the following steps:
(1) by phospholipid solution with being dried with nitrogen film method or rotary evaporation film legal system obtains immobilized artificial membrane, then by immobilized artificial membrane assembling
Liquid dissolution, obtains the immobilized artificial membrane assembles concentration；
(2) immobilized artificial membrane assembles concentration made from step (1) and the membrane scaffold proteins with affinity tag are mixed at 4-60 DEG C
Reaction 0.5-12h is closed, the molar ratio of membrane scaffold proteins and phosphatide is 1:(10-100 in immobilized artificial membrane assembles concentration), obtain charge type phosphorus
Rouge nanometer plate；
(3) charge type phosphatide nanometer plate made from step (2) is dialysed in coupling buffer to balance, then existed with affinity substrate
Coupling reaction 18-30h is carried out at 20-40 DEG C, obtains the phosphatide nanometer plate chromatography media.
10. phosphatide nanometer plate chromatography media according to any one of claims 1 to 5 answering in memebrane protein is purified and reconstructed
With；
Preferably, the method for the application are as follows: by the phosphatide nanometer plate chromatography media successively through dress column, balance, albumen loading,
Elution, elution and cleaning, realize the purifying and reconstruct to memebrane protein.
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| CN112108116A (en) * | 2020-09-21 | 2020-12-22 | 中国科学院新疆理化技术研究所 | Preparation method and testing device of formed carbon composite material capable of removing various pesticide residues and antibiotics simultaneously |
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| CN112108116A (en) * | 2020-09-21 | 2020-12-22 | 中国科学院新疆理化技术研究所 | Preparation method and testing device of formed carbon composite material capable of removing various pesticide residues and antibiotics simultaneously |
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