CN105294855B - A kind of preparation method of no phosphatide nanometer plate - Google Patents
A kind of preparation method of no phosphatide nanometer plate Download PDFInfo
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- CN105294855B CN105294855B CN201510824261.XA CN201510824261A CN105294855B CN 105294855 B CN105294855 B CN 105294855B CN 201510824261 A CN201510824261 A CN 201510824261A CN 105294855 B CN105294855 B CN 105294855B
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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Abstract
The present invention provides a kind of preparation method of no phosphatide nanometer plate, which comprises the steps of: Step 1: class phosphatide detergent is added in memebrane protein purification phase；Step 2: memebrane protein skelemin is added, is mixed with vortex mixer, the constant-temperature incubation predetermined time completing memebrane protein after purification；Step 3: being concentrated after molecular sieve and affinitive layer purification, products therefrom is collected.Method of the invention eliminates in conventional method, and phosphatide is dissolved in detergent, memebrane protein skelemin is proportionally mixed and utilized dialysis or hydrophobic adsorbent removal these three steps of detergent with the phosphatide being first dissolved in detergent in advance.So that the preparation step without phosphatide nanometer plate greatly simplifies, while also avoiding the loss of memebrane protein.
Description
Technical field
The present invention relates to a kind of preparation methods of no phosphatide nanometer plate, belong to field of biotechnology.
Background technique
Memebrane protein is the general name for referring to combine or be integrated into cell membrane or the protein on organelle film.All known
In albumen, memebrane protein almost occupies 40%.Including receptor, channel protein, signaling molecule is necessary to cell communications,
Its rich content in cell, memebrane protein are the main executives of biomembrane function, in substance transportation, the side such as cellular informatics identification
Exercise critical function in face；If memebrane protein disabler will lead to many diseases.In the drug target albumen being currently known,
60% is memebrane protein [Wallin E, Von Heijne G.Genome-wide analysis of integral membrane
proteins from eubacterial,archaean,and eukaryotic organisms.Protein Sci.1998；
7:1029–1038；Czerski L.,Sanders C.R.Anal.Biochem.2000；284:327.；Hu,Y.,Y.Li,
X.Zhang,X.Guo,B.Xia,and C.Jin.2006.Solution structures and backbone dynamics
of a flavodoxin MioC from Escherichia coli in both apo-and holo-
forms.J.Biol.Chem.281:35454–35466.]。
In vitro, memebrane protein needs to maintain lipid bilayer or lipoid environment that can just keep its activated state, this is for film
The structure and function research of albumen, the exploitation of drug and screening are all and its important.But it is difficult to simulate this environment in vitro.
Phosphatide nanometer plate (Nanodisc) is the novel class for memebrane protein research developed by high-density lipoprotein
Membrane structure, which stablizes phospholipid molecule using amphiphilic albumen in water phase, to form the class cell membrane of Lipid bilayer membranes
Structure, bioactivity can be kept in approximate natural membrane environment by being integrated into memebrane protein therein, thus for further research
Provide platform.When assembling phosphatide nanometer plate (Nanodisc), first by memebrane protein skelemin (Membrane Scaffold
Protein, MSP) proportionally mixed with the phosphatide being first dissolved in detergent (usually sodium taurocholate) in advance.It is incubated through certain time
It educates, detergent is removed using dialysis or hydrophobic adsorbent, such as Bio-Beads SM-2 (Bio-Rad, Hercules, CA), from group
Dress forms phosphatide nanometer plate (Nanodisc) [Nath A., Atkins W.M., Sligar S.G.Applications of
phospholipid bilayer nanodiscs in the study of membranes and membrane
proteins.Biochemistry.2007；46:2059–2069.].Assembling includes the phosphatide nanometer plate of memebrane protein
(Nanodisc) appropriate memebrane protein need to be only added in system.Ionophorous protein, GPCRs, transport protein and cytochromes
The memebrane proteins such as P450, which successfully assemble the system also with phosphatide nanometer plate (Nanodisc), has solvable, stable and size
The advantages that controllable.Therefore, phosphatide nanometer plate (Nanodisc) system will become the important carrier of memebrane protein research.
However traditional preparation method assembling steps are comparatively laborious, and will cause the loss of protein.
Summary of the invention
The purpose of the present invention is to provide a kind of preparation methods of no phosphatide nanometer plate, to solve the above problems.
Present invention employs following technical solutions:
A kind of preparation method of no phosphatide nanometer plate, which comprises the steps of:
Step 1: class phosphatide detergent is added in memebrane protein purification phase；
Step 2: memebrane protein skelemin is added, is mixed with vortex mixer, constant-temperature incubation is pre- completing memebrane protein after purification
It fixes time；
Step 3: being concentrated after molecular sieve and affinitive layer purification, products therefrom is collected.
Further, the preparation method of no phosphatide nanometer plate of the invention, can also have the following features: wherein, described
Memebrane protein skelemin is Apolipoprotein A-1, and amino acid sequence is as shown in SEQ ID NO.1, encoding gene sequence
Column are as shown in SEQ ID NO.2.
Further, the preparation method of no phosphatide nanometer plate of the invention, can also have the following features: wherein, described
Class phosphatide detergent is FOS-CHOLINE, and C chain contains 8-16 C.
Further, the preparation method of no phosphatide nanometer plate of the invention, can also have the following features: wherein, described
The molar ratio of memebrane protein and the memebrane protein skelemin is 1:2-1:20.
Further, the preparation method of no phosphatide nanometer plate of the invention, can also have the following features: wherein, described
The temperature of constant-temperature incubation is 0-37 DEG C.
Preferably, the preparation method of no phosphatide nanometer plate of the invention, can also have the following features: wherein, it is described
The temperature of constant-temperature incubation is 4-20 DEG C.
Further, the preparation method of no phosphatide nanometer plate of the invention, can also have the following features: wherein, described
Predetermined time is 1-6h.
Advantageous effect of the invention
The preparation method of no phosphatide nanometer plate according to the present invention, due to directlying adopt class phosphatide detergent purification film egg
It is white, memebrane protein skelemin is then added after purification in memebrane protein, therefore eliminate phosphatide nanometer plate (Nanodisc) and prepared
Phosphatide is dissolved in detergent (usually sodium taurocholate) in advance in journey, by memebrane protein skelemin (Membrane Scaffold
Protein, MSP) dialysis is proportionally mixed and utilized with the phosphatide being first dissolved in detergent (usually sodium taurocholate) in advance
Or hydrophobic adsorbent, as Bio-Beads SM-2 (Bio-Rad, Hercules, CA) removes these three steps of detergent.So that nothing
The preparation step of phosphatide nanometer plate greatly simplifies, while also avoiding the loss of memebrane protein.
Detailed description of the invention
Fig. 1 is the electromicroscopic photograph without phosphatide nanometer plate that embodiment three obtains.
Specific embodiment
In order to which those skilled in the art better understood when technical solution provided by the present invention, below with reference to specific
Embodiment is illustrated.Reagent used in following embodiment then can be purchased unless otherwise instructed by disclosed commercial channel
It buys and obtains.Experimental method used in following embodiment unless otherwise instructed, then according to the common side of experiment of biology
Method carries out.
Embodiment one
Class phosphatide detergent Fos-Choline-8 is added in memebrane protein purification phase, completes memebrane protein after purification, is being added
Memebrane protein skelemin Membrane Scaffold Protein (MSP).Memebrane protein and memebrane protein skelemin in reaction solution
Molar ratio be 1:2, mixed with vortex mixer, 4 DEG C of constant-temperature incubation 1h are concentrated after molecular sieve and affinitive layer purification, collect institute
Product is obtained, passes through transmission electron microscope observing products therefrom and disc-shaped structure, diameter 5-10nm is presented.The carbochain of Fos-Choline-8
Contain 8 C.Memebrane protein, which purifies the cell used, two classes, is respectively: insect cell: such as sf9 High Five；Or
Person's mammalian cell: such as 293F.These types of cell can lead to commercial channel and be commercially available.It is of course also possible to use this field
Source of the cell of common other types as memebrane protein.The carbochain of Fos-Choline-14 contains 14 C.
Embodiment two
Class phosphatide detergent Fos-Choline-10 is added in memebrane protein purification phase, completes memebrane protein after purification, is adding
Enter memebrane protein skelemin.Molar ratio is 1:6, is mixed with vortex mixer, 8 DEG C of constant-temperature incubation 2h, pure through molecular sieve and affinity chromatography
It is concentrated after change, collects products therefrom, disc-shaped structure, diameter 5-10nm are presented by transmission electron microscope observing products therefrom.Fos-
The carbochain of Choline-10 contains 10 C.
Embodiment three
Class phosphatide detergent Fos-Choline-14 is added in memebrane protein purification phase, completes memebrane protein after purification, is adding
Enter memebrane protein skelemin.The molar ratio of memebrane protein and memebrane protein skelemin is 1:8, is mixed with vortex mixer, 4 DEG C of constant-temperature incubations
2h is concentrated after molecular sieve and affinitive layer purification, collects products therefrom.Disk is presented by transmission electron microscope observing products therefrom
Shape structure, diameter 5-10nm.As shown in Figure 1, being the nanometer plate being prepared using present embodiment at arrow A meaning in figure
One of.
Example IV
Class phosphatide detergent Fos-Choline-12 is added in memebrane protein purification phase, completes memebrane protein after purification, is adding
Enter memebrane protein skelemin.Molar ratio is 1:10, is mixed with vortex mixer, 10 DEG C of constant-temperature incubation 3h, through molecular sieve and affinity chromatography
It is concentrated after purification, collects products therefrom, disc-shaped structure, diameter 5-10nm are presented by transmission electron microscope observing products therefrom.
The carbochain of Fos-Choline-12 contains 12 C.
Embodiment five
Class phosphatide detergent Fos-Choline-14 is added in memebrane protein purification phase, completes memebrane protein after purification, is adding
Enter memebrane protein skelemin (Membrane Scaffold Protein, MSP).Molar ratio is 1:12, is mixed with vortex mixer, 12
DEG C constant-temperature incubation 6h, is concentrated after molecular sieve and affinitive layer purification, collects products therefrom, the production as transmission electron microscope observing obtained by
Disc-shaped structure, diameter 5-10nm is presented in object.The carbochain of Fos-Choline-14 contains 14 C.
Embodiment six
Class phosphatide detergent Fos-Choline-16 is added in memebrane protein purification phase, completes memebrane protein after purification, is adding
Enter memebrane protein skelemin (Membrane Scaffold Protein, MSP).Molar ratio is 1:16, is mixed with vortex mixer, 16
DEG C constant-temperature incubation 5h, is concentrated after molecular sieve and affinitive layer purification, collects products therefrom, the production as transmission electron microscope observing obtained by
Disc-shaped structure, diameter 5-10nm is presented in object.The carbochain of Fos-Choline-16 contains 16 C.
Embodiment seven
Class phosphatide detergent Fos-Choline-14 is added in memebrane protein purification phase, completes memebrane protein after purification, is adding
Enter memebrane protein skelemin (Membrane Scaffold Protein, MSP).Molar ratio is 1:20, is mixed with vortex mixer, 20
DEG C constant-temperature incubation 4h, is concentrated after molecular sieve and affinitive layer purification, collects products therefrom, the production as transmission electron microscope observing obtained by
Disc-shaped structure, diameter 5-10nm is presented in object.The carbochain of Fos-Choline-14 contains 14 C.
In the above various embodiments, Apolipoprotein A-1 refers to: aPoA-I.
FOS-CHOLINE refers to: FOS-CHOLIN is a trade name, is the general designation of this kind of detergents.Wherein, FOS-
CHOLINE-12: dodecylphosphoric acid choline；FOS-CHOLINE-8: octylphosphonic acid choline；FOS-CHOLINE-14: the tetradecane
Base phosphocholine；FOS-CHOLINE-10 decyl choline phosphate.
Claims (6)
1. a kind of preparation method of no phosphatide nanometer plate, which comprises the steps of:
Step 1: class phosphatide detergent is added in memebrane protein purification phase,
Step 2: memebrane protein skelemin is added, is mixed with vortex mixer, the pre- timing of constant-temperature incubation completing memebrane protein after purification
Between；
Step 3: being concentrated after molecular sieve and affinitive layer purification, products therefrom is collected,
Do not include wherein: phosphatide being dissolved in detergent in advance, by memebrane protein skelemin proportionally be first dissolved in advance
Phosphatide in detergent mixes and removes these three steps of detergent using dialysis or hydrophobic adsorbent.
2. the preparation method as described in claim 1 without phosphatide nanometer plate, it is characterised in that:
Wherein, the memebrane protein skelemin be ApolipoproteinA-1, amino acid sequence as shown in SEQ ID NO.1,
Its coding gene sequence is as shown in SEQ ID NO.2.
3. the preparation method as described in claim 1 without phosphatide nanometer plate, it is characterised in that:
Wherein, the molar ratio of the memebrane protein and the memebrane protein skelemin is 1:2-1:20.
4. the preparation method as described in claim 1 without phosphatide nanometer plate, it is characterised in that:
Wherein, the temperature of the constant-temperature incubation is 0-37 DEG C.
5. the preparation method as claimed in claim 4 without phosphatide nanometer plate, it is characterised in that:
Wherein, the temperature of the constant-temperature incubation is 4-20 DEG C.
6. the preparation method as described in claim 1 without phosphatide nanometer plate, it is characterised in that: wherein, the predetermined time is
1-6h。
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| CN109276570B (en) * | 2018-10-31 | 2020-09-25 | 西北工业大学 | Nano medicine of biological source macrocyclic molecule and its preparing method |
| CN114940711B (en) * | 2021-10-25 | 2023-05-12 | 中山大学 | Apolipoprotein A-I mimetic peptides and uses thereof |
Citations (3)
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| CN1909888A (en) * | 2004-01-13 | 2007-02-07 | 伊利诺斯州立大学托管会 | Membrane scaffold proteins |
| CN101128586A (en) * | 2004-12-22 | 2008-02-20 | 健泰科生物技术公司 | Methods for producing soluble membrane-spanning proteins |
| CN104655737A (en) * | 2013-11-18 | 2015-05-27 | 维亚生物科技(上海)有限公司 | Method for determining affinity of membrane protein and ligand based on liquid chromatography tandem mass spectrometry |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US8999320B2 (en) * | 2008-01-30 | 2015-04-07 | The Rockefeller University | Nanoscale bound bilayers, methods of use and production |
| US9644038B2 (en) * | 2011-12-21 | 2017-05-09 | The Regents Of The University Of California | Apolipoprotein nanodiscs with telodendrimer |
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Patent Citations (3)
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| CN1909888A (en) * | 2004-01-13 | 2007-02-07 | 伊利诺斯州立大学托管会 | Membrane scaffold proteins |
| CN101128586A (en) * | 2004-12-22 | 2008-02-20 | 健泰科生物技术公司 | Methods for producing soluble membrane-spanning proteins |
| CN104655737A (en) * | 2013-11-18 | 2015-05-27 | 维亚生物科技(上海)有限公司 | Method for determining affinity of membrane protein and ligand based on liquid chromatography tandem mass spectrometry |
Non-Patent Citations (2)
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| Detergent/Nanodisc Screening for High-Resolution NMR Studies of an Integral Membrane Protein Containing a Cytoplasmic Domain;Christos Tzitzilonis et al.;《PLOS ONE》;20130122;第8卷(第1期);第2-3页材料和方法部分，第3页左栏第3段 * |
| Structures of the OmpF porin crystallized in the presence of foscholine-12;Georgia Kefala et al.;《PROTEIN SCIENCE》;20100301;第19卷(第5期);1117-1125 * |
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