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Membrane scaffold protein, phospholipid nanodisk and nanoparticle as well as preparation methods thereof

A technology of phospholipid nanodiscs and membrane scaffolds, applied in chemical instruments and methods, nanomedicine, nanotechnology, etc., can solve the problems of large activity loss and achieve high success rate, large protein activity, and small batch-to-batch difference

Active Publication Date: 2021-03-26
CUSABIO TECH LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are many ways to use these membrane scaffold proteins to stabilize, disperse and solubilize fully or partially hydrophobic proteins (such as peripheral, embedding or integral membrane proteins) while maintaining the biological activity of these membrane proteins, or to stabilize, disperse and solubilize membrane fragments However, when the phospholipid nanodisks formed by membrane scaffold proteins in the prior art are used to stabilize, disperse and dissolve completely or partially hydrophobic proteins, the activity loss is large. Taking plasma membrane proteins as an example, after a series of purification steps, the protein activity Only about 30%, and the refactoring process only increased its activity to 60% at best

Method used

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  • Membrane scaffold protein, phospholipid nanodisk and nanoparticle as well as preparation methods thereof
  • Membrane scaffold protein, phospholipid nanodisk and nanoparticle as well as preparation methods thereof
  • Membrane scaffold protein, phospholipid nanodisk and nanoparticle as well as preparation methods thereof

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preparation example Construction

[0044] According to another typical implementation of the embodiments of the present invention, a method for preparing the membrane scaffold protein is provided, the method comprising:

[0045] Obtain a gene fragment encoding the amino acid sequence shown in SEQ ID NO.1;

[0046] The gene fragment is cloned between the NcoI and HindIII restriction sites of the pET28a vector to obtain the recombinant vector pET28a-MPS1D1;

[0047] After transforming the recombinant vector pET28a-MPS1D1, inducing expression and purifying, the membrane scaffold protein is obtained.

[0048] According to another typical implementation of the embodiments of the present invention, there is provided a phospholipid nanodisk, the preparation raw materials of the phospholipid nanodisk include membrane scaffold protein solution, lipid solution and detergent DPC; wherein, the membrane scaffold The protein solution is a solution obtained by mixing the membrane scaffold protein with a buffer; the lipid sol...

Embodiment 1

[0076] Example 1 Preparation of recombinant vector pET28a-MPS1D1

[0077] 1. The amino acid sequence of the membrane scaffold protein MPS1D1 is shown in SEQ ID NO.1.

[0078] 2. Acquisition of Membrane Scaffold Protein MPS1D1 Gene Fragment

[0079] The gene fragments were synthesized by Wuhan Jinkairui Bioengineering Co., Ltd. Primers were designed, and the primers are shown in Table 1.

[0080] Table 1

[0081]

[0082] Using the synthesized gene fragment as a template, the primer pair shown in SEQ ID NO.3-4 was used to carry out PCR reaction, and the reaction system is shown in Table 2.

[0083] Table 2

[0084] project volume template 4μL Upstream primer (F) 2μL Downstream primer (R) 2μL 10×Buffer 20 μL dNTPs 16μL pFu enzyme 2μL Add water to 200μL

[0085] The PCR reaction program was: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 30 sec, annealing at 52°C for 45 sec, and extension at 72...

Embodiment 2

[0102] Example 2 Expression and purification of membrane scaffold protein MPS1D1 protein

[0103] 1. Expression of vector pET28a-MPS1D1 protein

[0104] Prepare the Escherichia coli expression host strain Rosetta (DE3) and melt it on ice, and add the pET28a-MSP1D1 plasmid. Place on ice for 30 minutes; heat shock at 42°C for 90s, place on ice for 1 minute, add 800 μL of preheated LB medium, and place in a shaker at 37°C for 158r / 120min. Centrifuge at 6000r for 4min, absorb 800μL supernatant in the ultra-clean bench, mix the remaining bacterial solution, and spread it on a plate containing K+; invert the plate, and culture it overnight in a constant temperature incubator at 37°C.

[0105] Pick a single colony and add it to a LB (3mL) test tube corresponding to the resistance, put it in a shaker and culture it for 3h, take 700μL of the suspension and add it to 100μL (C=50%) of seed-preserving glycerol, shake well, and put it in the refrigerator ( -20°C) for freezing. Then add ...

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Abstract

In the embodiment, the invention provides a membrane scaffold protein, a phospholipid nanodisk and a nanoparticle as well as preparation methods thereof. The membrane scaffold protein has an amino acid sequence shown in SEQ ID NO. 1; the phospholipid nanodisk is a discoid phospholipid bilayer, and is prepared from the following raw materials: the membrane scaffold protein, a lipid solution and a buffer solution, wherein the lipid solution is prepared by dissolving lipid with a 50-100 mM sodium cholate solution until the final concentration of 45-55 mM is reached; and the nanoparticle is prepared from the following raw materials: the phospholipid nanodisk, and membrane protein. According to the embodiment of the invention, by preparing the nanoparticle from the phospholipid nanodisk and themembrane protein, relatively high protein activity can be kept for the membrane protein, thereby having related biological functions of the membrane protein retained; and thus, the membrane protein purified by the method has high purity and high activity.

Description

technical field [0001] The embodiment of the invention belongs to the field of biotechnology, and relates to a membrane scaffold protein, a phospholipid nanodisk, a nanoparticle and a preparation method thereof. Background technique [0002] As far as the development of new biological drugs is concerned, membrane proteins are important targets, and GPCRs, ion channel proteins, transporters, etc. play an important role in intercellular communication, receiving external signals, and receiving other substances. In recent years, membrane protein research has made significant progress, which is inseparable from the advancement of related tools and reagents for membrane protein research. For a more in-depth study of the structure and function of membrane proteins, it is crucial to correctly extract and assemble membrane proteins into a membrane-like environment. The application of nanodisc technology can effectively improve the stability of membrane proteins in the in vitro envir...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/00C12N15/70C12N15/66C07K14/245B82Y5/00B82Y40/00
CPCC07K14/001C12N15/70C07K14/245C07K19/00B82Y5/00B82Y40/00
Inventor 王静易汪雪孙柯汤碰
Owner CUSABIO TECH LLC
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