Preparation method of synaptic vesicle protein SV2A

Abstract

The invention provides a preparation method of synaptic vesicle protein SV2A. The preparation method comprises the following steps: 1) overexpression: transferring the 104th-742nd basic groups of a human SV2A gene with a label for protein affinity purification into an animal cell through a virus vector to overexpress the animal cell; (2) dissolving: cracking the animal cell, and dissolving membrane protein by using an excessive mixed solution of n-dodecyl beta-D-maltitoside and cholesterol hemicellulose succinate relative to the membrane protein, wherein the mass concentration ratio of the n-dodecyl beta-D-maltitoside to the cholesterol hemicellulose succinate is 10: 1; and 3) purification: performing centrifuging, taking supernate, performing purifying by affinity chromatography, replacing n-dodecyl beta-D-maltitoside and cholesterol hemicellulose succinate trisalt in the protein solution by using a digitonin solution which is excessive relative to the membrane protein, and performing purifying again by using a molecular sieve to obtain the SV2A protein. According to the method, the SV2A protein with stable configuration can be successfully separated.

Description

technical field [0001] The invention relates to the field of membrane protein expression and purification. Background technique [0002] Synaptic vesicle protein SV2A is a class of glycoproteins with 12 transmembrane structures present in all synaptic and endocrine vesicles. SV2A is also the target molecule of the antiepileptic drug levetiracetam. However, the specific function of SV2A is unclear. Large-scale preparation of SV2A is helpful for the study of its structure and function, and for the development of related drugs. [0003] The preparation of membrane proteins usually requires four main steps: overexpression in the host system, dissolution of cell membranes with detergents, protein purification, and Nanodisc (phospholipid nanodisc) assembly. [0004] Among them, overexpression is the biggest bottleneck in the membrane protein preparation process. The choice of host system depends on the requirements for membrane proteins (such as post-translational modification,...

AI Technical Summary

Problems solved by technology

[0007] Due to the complex structure of SV2A protein, researchers often encounter protein high aggregation when expressing and purifying the protein in vitro, so that the obtained SV2A cannot exist stably in the inherent conformation of the membrane protein. The conformation is very different, theoretically it does not have the biological function of the membrane protein

[0008] So far, there has been no report on the expression of SV2A protein with a stable conformation of membrane protein in vitro

Images