CN105294855A - Preparation method of non-phospholipid nanodisc - Google Patents
Preparation method of non-phospholipid nanodisc Download PDFInfo
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- CN105294855A CN105294855A CN201510824261.XA CN201510824261A CN105294855A CN 105294855 A CN105294855 A CN 105294855A CN 201510824261 A CN201510824261 A CN 201510824261A CN 105294855 A CN105294855 A CN 105294855A
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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 invention provides a preparation method of a non-phospholipid nanodisc, and is a characterized in that the preparation method includes the following steps: step one, in a membrane protein purification stage, adding a phospholipid-like detergent; step two, after completion of membrane protein purification, adding a membrane scaffold protein, mixing evenly with a uniform mixing device, and carrying out thermostatic incubation for a scheduled time; and step three, after purifying with molecular sieve and affinity chromatography, concentrating, and collecting the product. The method omits three steps in a traditional method of dissolving phospholipid in a detergent, mixing a membrane scaffold protein with phospholipid pre-dissolved in the detergent according to the proportion and removing the detergent with dialysis or a hydrophobic adsorbent. Therefore, the preparation steps of the non-phospholipid nanodisc are greatly simplified, and besides, the loss of membrane proteins is avoided.
Description
Technical field
The present invention relates to a kind of preparation method without phosphatide nanometer plate, belong to biological technical field.
Background technology
Membranin refer to can in conjunction with or be incorporated into the general name of the protein on cytolemma or organoid film.In all known albumen, membranin almost occupies 40%.Comprise acceptor, channel protein, signaling molecule, be that cell communications is necessary, it is rich content in cell, and membranin is the main executive of microbial film function, and at substance transportation, critical function is exercised in the aspects such as cellular informatics identification; If membranin disabler, then can cause a lot of disease.In drug target albumen known at present, 60% is membranin [WallinE, VonHeijneG.Genome-wideanalysisofintegralmembraneproteins fromeubacterial, archaean, andeukaryoticorganisms.ProteinSci.1998; 7:1029 – 1038; CzerskiL., SandersC.R.Anal.Biochem.2000; 284:327.; Hu, Y., Y.Li, X.Zhang, X.Guo, B.Xia, andC.Jin.2006.Solutionstructuresandbackbonedynamicsofafl avodoxinMioCfromEscherichiacoliinbothapo-andholo-forms.J .Biol.Chem.281:35454 – 35466.].
In vitro, membranin needs to maintain lipid bilayer or lipoid environment just can keep its active condition, and this structure and function for membranin study, the exploitation of medicine and screen and be all and important.But be difficult to this environment of simulation in vitro.
Phosphatide nanometer plate (Nanodisc) is the novel membranelike structure studied for membranin developed by high-density lipoprotein (HDL), this system adopts amphiphilic albumen to stablize phospholipid molecule in aqueous phase, thus form the class membrane structure of Lipid bilayer membranes, the membranin be integrated into wherein can keep biological activity in approximate natural membranes environment, thus provides platform for studying further.During assembling phosphatide nanometer plate (Nanodisc), first membranin skelemin (MembraneScaffoldProtein, MSP) is proportionally mixed with the phosphatide be dissolved in advance in stain remover (normally Sodium cholic acid).Hatch through certain hour, utilize dialysis or hydrophobic adsorbent, as Bio-BeadsSM-2 (Bio-Rad, Hercules, CA) stain remover is removed, self-assembly forms phosphatide nanometer plate (Nanodisc) [NathA., AtkinsW.M., SligarS.G.Applicationsofphospholipidbilayernanodiscsinth estudyofmembranesandmembraneproteins.Biochemistry.2007; 46:2059 – 2069.].The phosphatide nanometer plate (Nanodisc) that assembling comprises membranin only need add appropriate membranin again in system.Ionophorous protein, GPCRs, the membranin such as translocator and Cytochrome P450 successfully and phosphatide nanometer plate (Nanodisc) assemble this system and also have solvable, the advantages such as stable and size is controlled.Therefore, phosphatide nanometer plate (Nanodisc) system will become the important carrier of membranin research.
But traditional preparation method's number of assembling steps is more loaded down with trivial details, and the loss of protein can be caused.
Summary of the invention
The object of the present invention is to provide a kind of preparation method without phosphatide nanometer plate, to solve the problem.
Present invention employs following technical scheme:
Without a preparation method for phosphatide nanometer plate, it is characterized in that, comprise the steps:
Step one, add class phosphatide stain remover in membranin purification phase;
Step 2, after completing membranin purifying, add membranin skelemin, with vortex mixer mixing, the constant-temperature incubation scheduled time;
Step 3, concentrated after molecular sieve and affinitive layer purification, collect products therefrom.
Further, preparation method without phosphatide nanometer plate of the present invention, can also have such feature: wherein, and described membranin skelemin is ApolipoproteinA-1, its aminoacid sequence is as shown in SEQIDNO.1, and its coding gene sequence is as shown in SEQIDNO.2.
Further, the preparation method without phosphatide nanometer plate of the present invention, can also have such feature: wherein, and described class phosphatide stain remover is FOS-CHOLINE, and its C chain contains 8-16 C.
Further, the preparation method without phosphatide nanometer plate of the present invention, can also have such feature: wherein, and the mol ratio of described membranin and described membranin skelemin is 1:2-1:20.
Further, the preparation method without phosphatide nanometer plate of the present invention, can also have such feature: wherein, and the temperature of described constant-temperature incubation is 0-37 DEG C.
Preferably, the preparation method without phosphatide nanometer plate of the present invention, can also have such feature: wherein, and the temperature of described constant-temperature incubation is 4-20 DEG C.
Further, the preparation method without phosphatide nanometer plate of the present invention, can also have such feature: wherein, and the described scheduled time is 1-6h.
The beneficial effect of the invention
According to the preparation method without phosphatide nanometer plate of the present invention, owing to directly adopting class phosphatide stain remover purification membranin, then after membranin purifying, membranin skelemin is added, therefore eliminate in phosphatide nanometer plate (Nanodisc) preparation process and in advance phosphatide is dissolved in stain remover (normally Sodium cholic acid), by membranin skelemin (MembraneScaffoldProtein, MSP) proportionally mix with the phosphatide be dissolved in advance in stain remover (normally Sodium cholic acid) and utilize and dialyse or hydrophobic adsorbent, as Bio-BeadsSM-2 (Bio-Rad, Hercules, CA) these three steps of stain remover are removed.Preparation process without phosphatide nanometer plate is simplified greatly, also avoid the loss of membranin simultaneously.
Accompanying drawing explanation
Fig. 1 is the electromicroscopic photograph without phosphatide nanometer plate that embodiment three obtains.
Embodiment
In order to those skilled in the art can understand technical scheme provided by the present invention better, set forth below in conjunction with specific embodiment.The reagent that following embodiment is used, if no special instructions, then all buys by disclosed commercial channel and obtains.The experimental technique that following embodiment uses if no special instructions, then all carries out according to the experimental technique that biology is conventional.
Embodiment one
Add class phosphatide stain remover Fos-Choline-8 in membranin purification phase, after completing membranin purifying, add membranin skelemin MembraneScaffoldProtein (MSP).In reaction soln, the mol ratio of membranin and membranin skelemin is 1:2, with vortex mixer mixing, and 4 DEG C of constant-temperature incubation 1h, concentrated after molecular sieve and affinitive layer purification, collect products therefrom, present disc-shaped structure by transmission electron microscope observing products therefrom, diameter 5-10nm.The carbochain of Fos-Choline-8 contains 8 C.The cell used described in membranin purifying has two classes, respectively: insect cell: as sf9 or HighFive; Or mammalian cell: as 293F.This several cell all can lead to commercial channel purchase and obtain.Certainly, the cell of other kind that this area also can be used to commonly use is as the source of membranin.The carbochain of Fos-Choline-14 contains 14 C.
Embodiment two
Add class phosphatide stain remover Fos-Choline-10 in membranin purification phase, after completing membranin purifying, add membranin skelemin.Mol ratio is 1:6, with vortex mixer mixing, and 8 DEG C of constant-temperature incubation 2h, concentrated after molecular sieve and affinitive layer purification, collect products therefrom, present disc-shaped structure by transmission electron microscope observing products therefrom, diameter 5-10nm.The carbochain of Fos-Choline-10 contains 10 C.
Embodiment three
Add class phosphatide stain remover Fos-Choline-14 in membranin purification phase, after completing membranin purifying, add membranin skelemin.The mol ratio of membranin and membranin skelemin is 1:8, with vortex mixer mixing, and 4 DEG C of constant-temperature incubation 2h, concentrated after molecular sieve and affinitive layer purification, collect products therefrom.Disc-shaped structure is presented, diameter 5-10nm by transmission electron microscope observing products therefrom.As shown in Figure 1, in figure, arrow A indication place is one of nanometer plate using present embodiment to prepare.
Embodiment four
Add class phosphatide stain remover Fos-Choline-12 in membranin purification phase, after completing membranin purifying, add membranin skelemin.Mol ratio is 1:10, with vortex mixer mixing, and 10 DEG C of constant-temperature incubation 3h, concentrated after molecular sieve and affinitive layer purification, collect products therefrom, present disc-shaped structure by transmission electron microscope observing products therefrom, diameter 5-10nm.The carbochain of Fos-Choline-12 contains 12 C.
Embodiment five
Add class phosphatide stain remover Fos-Choline-14 in membranin purification phase, after completing membranin purifying, add membranin skelemin (MembraneScaffoldProtein, MSP).Mol ratio is 1:12, with vortex mixer mixing, and 12 DEG C of constant-temperature incubation 6h, concentrated after molecular sieve and affinitive layer purification, collect products therefrom, present disc-shaped structure by transmission electron microscope observing products therefrom, diameter 5-10nm.The carbochain of Fos-Choline-14 contains 14 C.
Embodiment six
Add class phosphatide stain remover Fos-Choline-16 in membranin purification phase, after completing membranin purifying, add membranin skelemin (MembraneScaffoldProtein, MSP).Mol ratio is 1:16, with vortex mixer mixing, and 16 DEG C of constant-temperature incubation 5h, concentrated after molecular sieve and affinitive layer purification, collect products therefrom, present disc-shaped structure by transmission electron microscope observing products therefrom, diameter 5-10nm.The carbochain of Fos-Choline-16 contains 16 C.
Embodiment seven
Add class phosphatide stain remover Fos-Choline-14 in membranin purification phase, after completing membranin purifying, add membranin skelemin (MembraneScaffoldProtein, MSP).Mol ratio is 1:20, with vortex mixer mixing, and 20 DEG C of constant-temperature incubation 4h, concentrated after molecular sieve and affinitive layer purification, collect products therefrom, present disc-shaped structure by transmission electron microscope observing products therefrom, diameter 5-10nm.The carbochain of Fos-Choline-14 contains 14 C.
Above in each embodiment, ApolipoproteinA-1 refers to: aPoA-I.
FOS-CHOLINE refers to: FOS-CHOLIN is a trade(brand)name, is the general designation of this class stain remover.Wherein, FOS-CHOLINE-12: dodecylphosphoric acid choline; FOS-CHOLINE-8: octylphosphonic acid choline; FOS-CHOLINE-14: Tetradecylphosphocholine; FOS-CHOLINE-10 decyl choline phosphate.
Claims (7)
1. without a preparation method for phosphatide nanometer plate, it is characterized in that, comprise the steps:
Step one, add class phosphatide stain remover in membranin purification phase;
Step 2, after completing membranin purifying, add membranin skelemin, with vortex mixer mixing, the constant-temperature incubation scheduled time;
Step 3, concentrated after molecular sieve and affinitive layer purification, collect products therefrom.
2., as claimed in claim 1 without the preparation method of phosphatide nanometer plate, it is characterized in that:
Wherein, described membranin skelemin is ApolipoproteinA-1, and its aminoacid sequence is as shown in SEQIDNO.1, and its coding gene sequence is as shown in SEQIDNO.2.
3., as claimed in claim 1 without the preparation method of phosphatide nanometer plate, it is characterized in that:
Wherein, described class phosphatide stain remover is FOS-CHOLINE, and its C chain contains 8-16 C.
4., as claimed in claim 1 without the preparation method of phosphatide nanometer plate, it is characterized in that:
Wherein, the mol ratio of described membranin and described membranin skelemin is 1:2-1:20.
5., as claimed in claim 1 without the preparation method of phosphatide nanometer plate, it is characterized in that:
Wherein, the temperature of described constant-temperature incubation is 0-37 DEG C.
6., as claimed in claim 5 without the preparation method of phosphatide nanometer plate, it is characterized in that:
Wherein, the temperature of described constant-temperature incubation is 4-20 DEG C.
7., as claimed in claim 1 without the preparation method of phosphatide nanometer plate, it is characterized in that:
Wherein, the described scheduled time is 1-6h.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109276570A (en) * | 2018-10-31 | 2019-01-29 | 西北工业大学 | The Nano medication and preparation method thereof of biological source macrocycle molecule |
| CN114940711A (en) * | 2021-10-25 | 2022-08-26 | 中山大学 | Apolipoprotein A-I mimic peptide and application thereof |
Citations (5)
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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 |
| US20110059159A1 (en) * | 2008-01-30 | 2011-03-10 | The Rockefeller University | Nanoscale bound bilayers, methods of use and production |
| US20130165636A1 (en) * | 2011-12-21 | 2013-06-27 | The Regents Of The University Of California | Apolipoprotein nanodiscs with telodendrimer |
| 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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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| US20110059159A1 (en) * | 2008-01-30 | 2011-03-10 | The Rockefeller University | Nanoscale bound bilayers, methods of use and production |
| US20130165636A1 (en) * | 2011-12-21 | 2013-06-27 | The Regents Of The University Of California | Apolipoprotein nanodiscs with telodendrimer |
| 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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| CHRISTOS TZITZILONIS ET AL.: "Detergent/Nanodisc Screening for High-Resolution NMR Studies of an Integral Membrane Protein Containing a Cytoplasmic Domain", 《PLOS ONE》 * |
| GEORGIA KEFALA ET AL.: "Structures of the OmpF porin crystallized in the presence of foscholine-12", 《PROTEIN SCIENCE》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109276570A (en) * | 2018-10-31 | 2019-01-29 | 西北工业大学 | The Nano medication and preparation method thereof of biological source macrocycle molecule |
| 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 |
| CN114940711A (en) * | 2021-10-25 | 2022-08-26 | 中山大学 | Apolipoprotein A-I mimic peptide and application thereof |
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| CN105294855B (en) | 2018-12-04 |
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