CN110305205A - It is a kind of for producing the preparation method of the nanometer disk of Membrane protein antigen - Google Patents
It is a kind of for producing the preparation method of the nanometer disk of Membrane protein antigen Download PDFInfo
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- CN110305205A CN110305205A CN201910623155.3A CN201910623155A CN110305205A CN 110305205 A CN110305205 A CN 110305205A CN 201910623155 A CN201910623155 A CN 201910623155A CN 110305205 A CN110305205 A CN 110305205A
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Abstract
The present invention provides the preparation method of the nanometer disk for producing Membrane protein antigen, include the following steps: to prepare lipid stoste in 25-100mM chloroform；Lipid stoste material is assigned in disposable Glass Culture Tubes, and dry with mild nitrogen stream in draught cupboard；Simultaneously rotary glass culture tube is tilted, is turning to obtain thin film at Glass Culture Tubes lower wall；Residual solvent is removed, Glass Culture Tubes are placed in vacuum in high-vacuum jar is overnight to obtain dry adipose membrane；Buffer containing sodium taurocholate is added in dry adipose membrane, cholate additional amount is twice of required concentration；Vortex-glass model culture tube heats under 60 DEG C of tap water, and carries out ultrasonic wave, until solution is fully transparent, and remains on tube wall without lipid；Membrane skeleton protein is added in the phosphatide dissolved with cholate, required lipid: protein ratio is obtained, incubation time is 15-30 minutes；Membrane protein antigen, or freeze-drying long term storage are mixed immediately.
Description
Technical field
The present invention relates to Membrane protein antigen production technical fields, and in particular, to a kind of for producing Membrane protein antigen
The preparation method of nanometer disk.
Background technique
Memebrane protein is the target spot of 50% or more clinical medicine in the market, and the malfunction and cardiovascular disease of memebrane protein are disliked
Property the major diseases such as tumour and mental disorder it is closely related, but at present memebrane protein biological medicament it is extremely rare, high quality antigen
Production is the main bottleneck of antibody research and development.Therefore the antigen PRODUCTION TRAITS of memebrane protein becomes one of current hotspot.
Three categories are basically divided into according to the difficulty or ease of memebrane protein preparation and the position being distributed in film, memebrane protein: external film
Albumen or peripheral membrane protein, inherent memebrane protein or integrated membrane protein and rouge anchorin.Wherein, integrated membrane protein accounts for film egg
The 70%~80% of white total amount, be mainly characterized by water-insoluble and in conjunction with film closely, therefore these memebrane proteins only make
It could be dissolved down from film with when stronger detergent-treatment.G protein coupled receptor (G protein-coupled
Receptors, GPCRs) family is memebrane protein family most huge in the mankind, they can regulate and control a variety of extensive physiology mistakes
Journey, and having in cell surface can be with the target spot of patent medicine, therefore is also one of the important target spot of numerous medicament research and developments.According to system
Meter, the drug sales volume for targeting GPCR account for the 27% of world market.
Since there are hydrophobic parts for memebrane protein, it is easy polymerization in vitro, once departing from its stable natural membranes ring
Border will influence even to lose its physiological function.In traditional Membrane protein antigen preparation method, people are usually using referred to as detergent
" soap " molecule carry out the Membrane protein antigen in stablizing solution, this will affect Membrane protein antigen functionality and structural intergrity simultaneously
Keep Membrane protein antigen not crystallizable.
So people need to find a kind of suitable method and prepare fully functional and structural integrity Membrane protein antigen, and
Extracellular environment can be steadily present in.
Summary of the invention
For overcome the deficiencies in the prior art, the present invention provides a kind of for producing the nanometer disk of Membrane protein antigen
Preparation.To solve in the prior art, Membrane protein antigen is easy polymerization in vitro, once departing from its stable natural membrane environment,
It will influence the defect for even losing its physiological function.
Technical scheme is as follows: for producing the preparation method of the nanometer disk of Membrane protein antigen, including it is as follows
Step: the reconstruction of Membrane protein antigen in phospholipid bilayer nanometer disk:
(1) lipid stoste is prepared in 25-100mM chloroform；
(2) the desired amount of lipid stoste material is assigned in disposable Glass Culture Tubes, and is used mildly in draught cupboard
Nitrogen stream is dry；Simultaneously rotary glass culture tube is tilted, is turning to obtain thin film at Glass Culture Tubes lower wall；Removal residual is molten
Glass Culture Tubes, are placed in that vacuum in high-vacuum jar is overnight to obtain dry adipose membrane by agent；
(3) buffer containing sodium taurocholate is added in dry adipose membrane, cholate additional amount is the two of required concentration
Times；Vortex-glass model culture tube heats under hot tap-water (about 60 DEG C), and carries out ultrasonic wave, until solution is fully transparent, and
There is no lipid to remain on tube wall；
(4) membrane skeleton protein is added in the phosphatide dissolved with cholate, obtain required lipid: protein ratio is incubated
Educating the time is 15-30 minutes；
(5) the disk reconstruct mixture prepared can mix Membrane protein antigen, or freeze-drying long term storage immediately.
The incubation of Palmitoyl Phosphatidylcholine (POPC) carries out on ice or at 4 DEG C.
The incubation of dimyristoyl phosphatidyl choline (DMPC) carries out at room temperature.
Being incubated at 37 DEG C for dipalmitoylphosphatidylcholine (DPPC) carries out.
For producing the preparation method of the nanometer disk of Membrane protein antigen, also packet following steps:
(1) expression of membrane skeleton protein: use pET expression system BL21-Gold (DE3) bacterial strain (Stratagene) as
Host expresses membrane skeleton protein；
(2) purifying of membrane skeleton protein: ni-sepharose purification membrane skeleton protein is used, and runs SDS-PAGE and checks and carry out electricity
Electrospray mass spectrometry analysis, and protein concentration is calculated, obtain membrane skeleton protein.
The final concentration of cholate is 12-40mM in step (3).
The use of nanometer disk can carry out the preparation and functional study of Membrane protein antigen in determining membrane environment, later
Crystallize Membrane protein antigen.By using nuclear magnetic resonance (NMR), X-ray crystal diffraction, Cryo electron microscopy, atomic force is aobvious
Micro mirror and other methods can get structure chart of the Membrane protein antigen on atomic level.
By the package of nanometer disk, Membrane protein antigen after purification is keeping space structure stable and biological activity
Meanwhile the original state on cell membrane of Membrane protein antigen will be also simulated, transmembrane domain is embedded in inside phospholipid bilayer, allows
The Membrane protein antigen for being purified out steadily exercises its normal function as general Membrane protein antigen.
The invention has the benefit that nanometer disk can make Membrane protein antigen, such as GPCRs antigen, in artificial environment
In and nanometer disk re-assembly, form similar natural membrane structure.Later, this to be assembled into nanometer disk
Membrane protein antigen can be purified in the case where no detergent with conventional chromatography method.Nanometer disk and memebrane protein
The production that the package assembly of antigen makes it possible to memebrane protein antibody is possibly realized in vitro, but also the research of antibody drug is no longer
It is limited.
To sum up, the use of nanometer disk can efficiently and leniently aid in the preparation and effectively of Membrane protein antigen
It solves the problems, such as to be stabilized in an in vitro environment after Membrane protein antigen extracts, to guarantee that Membrane protein antigen can be as natural
Cell membrane in maintain its conformation and biological function.
Detailed description of the invention:
Fig. 1 is of the present invention for producing the structural schematic diagram of the nanometer disk of Membrane protein antigen.
Specific embodiment
In order to make goal of the invention of the invention, technical solution and technical effect are more clearly understood, below with reference to specific reality
Applying mode, the present invention is described further.It should be understood that specific embodiment described herein and relevant drawings, be only used for solving
The present invention is released, is not intended to limit the present invention.
Referring to Fig.1, nanometer disk of the present invention be by membrane skeleton protein (membrane scaffold proteins,
MSPs) 2 and phospholipid molecule 1 constitute phospholipid bilayer membranelike structure.Membrane skeleton protein (MSPs) 2 is apolipoprotein (apo)
The reduction version of A-I, they surround lipid bilayer to form discoid structure.Most common phosphatide is two nutmegs
Phosphatidyl choline (DMPC) or Palmitoyl Phosphatidylcholine (POPC).Membrane skeleton protein and phosphatide are self-assembled into nanometer
Disk.Phosphatide includes a hydrophobic surface and hydrophilic surface outwardly towards internal rouge layer, this structure makes nanometer disk water-soluble
There is very high solubility in liquid, while Membrane protein antigen 3 can also be made to dissolve in the case where no detergent.
1, the expression of membrane skeleton protein
Use pET expression system BL21-Gold (DE3) bacterial strain (Stratagene) as host expresses membrane skeleton protein.
The specific method is as follows:
(1) it prepares starting culture: the 30mL LB culture medium containing 30mg/L kanamycins is individually inoculated with a bacterium
Strain.It is about 0.4-0.6 (5-6 hours usual) that OD600 value is incubated to by suspension at 37 DEG C, under conditions of 250rpm.At this point, bacterium
Liquid can be used immediately or be saved overnight at 4 DEG C.
(2) it prepares 2.5L TB culture medium and sterilizes；Setting shaking table parameter: 37 DEG C, 500rpm, air 3L/min；Work as temperature
When reaching 37 DEG C, 25 milligrams of kanamycins and a few drop defoaming agents are added into bacterium solution, are placed on shaking table later.
(3) OD600 value is checked per hour.When OD600 value reaches 2.5-3.0 (usually in 3-4 hours), 1mM is added
IPTG continues to be incubated for 3 hours.In general, OD600 value finally reaches 10-15 at this time.
(4) 10 minutes are centrifuged to harvest thallus with the revolving speed of 8000g.2.5L TB culture medium collects the weight of wet granular
Usually between 50 to 60 grams.
2, the purifying of membrane skeleton protein: ni-sepharose purification membrane skeleton protein is used.It is purified according to universal method, specific side
Method is as follows:
(1) nickel column (3.4~6cm) passes through the 0.1M NiSO4 of 1 volume (50mL), then passes through 100mL water.Use 250mL
40mM phosphate buffer, pH 7.4 balances pillar.
(2) thallus is resuspended with 200mL 20mM phosphate buffer (pH7.4).Phenylmethylsulfonyl fluoride (PMSF) is added extremely
Final concentration of 1mM.After being resuspended completely, Triton X-100 to final concentration of 1% is added.5 milligrams of deoxyribonucleases are added
I.It is ultrasonically treated (three 1 minute bouts), is centrifuged 30 points with the revolving speed of 30,000g.
(3) centrifuged supernatant is added on column.It should pay attention to ensuring that flow velocity is no more than 10mL/min (about 1mL/min
cm2).Pillar is washed with 250mL the following solution:
40mM Tris/HCl, 0.3M NaCl, 1%Triton X-100, pH 8.0；
40mM Tris/HCl, 0.3M NaCl, 50mM sodium taurocholate, 20mM amide, pH 8.0；
40mM Tris/HCl, 0.3M NaCl, 50mM imidazoles, pH 8.0；
(4) 40mM Tris/HCl is used, 0.3M NaCl, 0.4M imidazoles elutes membrane skeleton protein.Collect 10-14mL elution
Liquid checks protein content with Coomassie blue stain.Protein example is filtered using 0.22mm syringe filter, then plus
Enter 0.01%NaN3Stored sample.
(5) analyze sample: the logical operation SDS-PAGE of lipidated protein is checked and is carried out Electrospray Ionization Mass Spectrometry.Use 1mm
Quartz colorimetric utensil measures absorbance at 280nm, compares with standard buffer solution, and calculates protein concentration.If desired, will
It is concentrated into 4-10mg/mL.Membrane skeleton protein can store a couple of days at 4 DEG C.For long term storage, sample is freezed or frozen
It is dry, and stored under 20 DEG C or lower temperature.
(6) after purification, pillar is washed with the aqueous solution containing 50mM EDTA, and is balanced with 20% ethyl alcohol.
3. the reconstruction of Membrane protein antigen in phospholipid bilayer nanometer disk
Lipid stoste prepares in 25-100mM chloroform and is stored in 20 DEG C.Determine that stock solution is dense by Phosphate analysis
Degree.The desired amount of chloroform-lipid stoste material is assigned in disposable Glass Culture Tubes, and with mild nitrogen in draught cupboard
Flow drying；Pass through rotary tube keeps certain angle that can obtain thin film at wall under the tube simultaneously.Residual solvent is removed,
It is overnight that pipe is placed in vacuum in high-vacuum jar.Buffer containing sodium taurocholate is added in dry adipose membrane.In general, gallbladder
Hydrochlorate additional amount is twice of required concentration.Vortex pipe heats under hot tap-water (about 60 DEG C), and is carrying out ultrasonic wave,
Until solution is fully transparent, and remain on tube wall without lipid.Membrane skeleton protein is added to the phosphatide dissolved with cholate
In, obtain required lipid: protein ratio, it is ensured that the final concentration of cholate is 12-40mM in reconstruct mixture, if any must
It wants, supplements standard buffer solution or cholate stock solution.Mixture is incubated at a proper temperature, incubation temperature depends on institute
Lipid, time are 15 minutes or the longer time.Self assembly temperature should be close to the Tm of lipid used.The self assembly of POPC exists
It carries out on ice or at 4 DEG C, DMPC is carried out at room temperature, and DPPC is carried out at 37 DEG C.The disk reconstruct mixture prepared can be stood
Mix Membrane protein antigen, or freeze-drying long term storage.
The above content is a further detailed description of the present invention in conjunction with specific preferred embodiments, and it cannot be said that
Specific implementation of the invention is only limited to these instructions.For those of ordinary skill in the art to which the present invention belongs, exist
Under the premise of not departing from present inventive concept, architectural form cans be flexible and changeable, can be with derivative series product.It only makes several
Simple deduction or replace all shall be regarded as belonging to present invention scope of patent protection determined by the appended claims.
Claims (6)
1. the preparation method for the nanometer disk for producing Membrane protein antigen, which comprises the steps of: phospholipid bilayer
The reconstruction of Membrane protein antigen in nanometer disk:
(1) lipid stoste is prepared in 25-100mM chloroform；
(2) the desired amount of lipid stoste material is assigned in disposable Glass Culture Tubes, and with mild nitrogen in draught cupboard
Flow drying；Simultaneously rotary glass culture tube is tilted, is turning to obtain thin film at Glass Culture Tubes lower wall；Residual solvent is removed, it will
Glass Culture Tubes are placed in that vacuum in high-vacuum jar is overnight to obtain dry adipose membrane；
(3) buffer containing sodium taurocholate is added in dry adipose membrane, cholate additional amount is twice of required concentration；Whirlpool
Glass Culture Tubes are revolved, are heated under 60 DEG C of tap water, and carry out ultrasonic wave, until solution is fully transparent, and residual without lipid
It stays on tube wall；
(4) membrane skeleton protein is added in the phosphatide dissolved with cholate, obtains required lipid: protein ratio, when incubation
Between be 15-30 minutes；
(5) the disk reconstruct mixture prepared can mix Membrane protein antigen, or freeze-drying long term storage immediately.
2. according to claim 1 for producing the preparation method of the nanometer disk of Membrane protein antigen, which is characterized in that palm fibre
The incubation of palmitic acid acyl oleoyl phosphatidylcholine (POPC) carries out on ice or at 4 DEG C.
3. according to claim 1 for producing the preparation method of the nanometer disk of Membrane protein antigen, which is characterized in that two
The incubation of dimyristoylphosphatidycholine (DMPC) carries out at room temperature.
4. according to claim 1 for producing the preparation method of the nanometer disk of Membrane protein antigen, which is characterized in that two
Being incubated at 37 DEG C for palmitoylphosphatidyl choline (DPPC) carries out.
5. according to claim 1 for producing the preparation method of the nanometer disk of Membrane protein antigen, which is characterized in that also
Packet following steps:
(1) expression of membrane skeleton protein: use pET expression system BL21-Gold (DE3) bacterial strain (Stratagene) as host
Express membrane skeleton protein；
(2) purifying of membrane skeleton protein: ni-sepharose purification membrane skeleton protein is used, and runs SDS-PAGE and checks and carry out electron spray
Mass spectral analysis, and protein concentration is calculated, obtain membrane skeleton protein.
6. according to claim 1 for producing the preparation method of the nanometer disk of Membrane protein antigen, which is characterized in that step
Suddenly the final concentration of cholate is 12-40mM in (3).
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112552378A (en) * | 2020-12-14 | 2021-03-26 | 武汉华美生物工程有限公司 | Membrane scaffold protein, phospholipid nanodisk and nanoparticle and preparation method thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1474831A (en) * | 2000-11-20 | 2004-02-11 | ����ŵ˹������ѧ�йܻ� | Membrane scaffold proteins |
| CN1909888A (en) * | 2004-01-13 | 2007-02-07 | 伊利诺斯州立大学托管会 | Membrane scaffold proteins |
| CN107090030A (en) * | 2017-05-31 | 2017-08-25 | 中国科学院合肥物质科学研究院 | Containing FGFR, phospholipid bilayer and membrane skeleton protein MSP class film system and preparation method thereof |
| US20190154698A1 (en) * | 2017-11-22 | 2019-05-23 | The Regents Of The University Of Michigan | Polymer-Based Lipid Nanodiscs And Macrodiscs |
-
2019
- 2019-07-11 CN CN201910623155.3A patent/CN110305205A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1474831A (en) * | 2000-11-20 | 2004-02-11 | ����ŵ˹������ѧ�йܻ� | Membrane scaffold proteins |
| CN1909888A (en) * | 2004-01-13 | 2007-02-07 | 伊利诺斯州立大学托管会 | Membrane scaffold proteins |
| CN107090030A (en) * | 2017-05-31 | 2017-08-25 | 中国科学院合肥物质科学研究院 | Containing FGFR, phospholipid bilayer and membrane skeleton protein MSP class film system and preparation method thereof |
| US20190154698A1 (en) * | 2017-11-22 | 2019-05-23 | The Regents Of The University Of Michigan | Polymer-Based Lipid Nanodiscs And Macrodiscs |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112552378A (en) * | 2020-12-14 | 2021-03-26 | 武汉华美生物工程有限公司 | Membrane scaffold protein, phospholipid nanodisk and nanoparticle and preparation method thereof |
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