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Preparation method of non-phospholipid nanodisc

A nanodisk and phospholipid-free technology, applied in the biological field, can solve the problems of cumbersome assembly steps and protein loss, and achieve the effect of simplifying the preparation steps and avoiding losses

Active Publication Date: 2016-02-03
VIVA BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the assembly steps of the traditional preparation method are cumbersome and cause protein loss

Method used

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  • Preparation method of non-phospholipid nanodisc
  • Preparation method of non-phospholipid nanodisc
  • Preparation method of non-phospholipid nanodisc

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Phospholipid detergent Fos-Choline-8 was added in the membrane protein purification stage, and membrane protein skeleton protein Membrane Scaffold Protein (MSP) was added after membrane protein purification was completed. The molar ratio of membrane protein and membrane protein skeleton protein in the reaction solution is 1:2, mix well with a mixer, incubate at a constant temperature at 4°C for 1 h, purify by molecular sieve and affinity chromatography, concentrate, collect the obtained product, and observe the obtained product by transmission electron microscope The product presents a disk-like structure with a diameter of 5-10 nm. The carbon chain of Fos-Choline-8 contains 8 Cs. There are two types of cells used in membrane protein purification, namely: insect cells: such as sf9 or HighFive; or mammalian cells: such as 293F. These types of cells are commercially available. Of course, other types of cells commonly used in the art can also be used as the source of mem...

Embodiment 2

[0025] Add the phospholipid detergent Fos-Choline-10 in the membrane protein purification stage, and add the membrane protein skeleton protein after the membrane protein purification is completed. The molar ratio is 1:6, mixed with a mixer, incubated at 8°C for 2 hours, purified by molecular sieves and affinity chromatography, concentrated, collected the product, and observed by transmission electron microscope. The obtained product presents a disc-shaped structure with a diameter of 5-10 nm . The carbon chain of Fos-Choline-10 contains 10 Cs.

Embodiment 3

[0027] Add the phospholipid detergent Fos-Choline-14 in the membrane protein purification stage, and add the membrane protein skeleton protein after the membrane protein purification is completed. The molar ratio of membrane protein and membrane protein skeleton protein was 1:8, mixed with a mixer, incubated at 4°C for 2 hours, purified by molecular sieves and affinity chromatography, concentrated, and the resulting product was collected. Observation by transmission electron microscope shows that the obtained product presents a disc-shaped structure with a diameter of 5-10 nm. Such as figure 1 As shown, the place indicated by the arrow A in the figure is one of the nanodiscs prepared by using this embodiment.

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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 [0001] The invention relates to a method for preparing a phospholipid-free nano-disc, which belongs to the field of biotechnology. Background technique [0002] Membrane proteins refer to the general term for proteins that can bind or integrate into cell membranes or organelle membranes. Membrane proteins account for almost 40% of all known proteins. Including receptors, channel proteins, and signaling molecules, which are necessary for cell communication and are abundant in cells. Membrane proteins are the main executors of biomembrane functions, and perform important functions in material transport, cell information recognition, etc.; if the membrane Failure of protein function can lead to many diseases. Among the currently known drug target proteins, 60% are membrane proteins [WallinE, VonHeijneG. Genome-wideanalysisofintegralmembraneproteinsfromeubacterial, archaean, andeukaryoticorganisms. ProteinSci. ;284:327.;Hu, Y., Y. Li, X. Zhang, X. Guo, B. Xia,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/705C07K1/22
CPCC07K14/705
Inventor 沈坚郭政李卫华金美辰苏晓燕韩敏李子娟钱冬明蔡建华任德林毛晨
Owner VIVA BIOTECH