High yield and efficient expression and purification of the human 5-HT3A receptor.

ABSTRACT:

Aim

To establish a method for efficient expression and purification of the human serotonin type 3A receptor (5-HT3A) that is suitable for structural studies.

Methods

Codon-optimized cDNA of human 5-HT3A was inserted into a modified BacMam vector, which contained an IgG leader sequence, an 8×His tag linked with two-Maltose Binding Proteins (MBP), and a TEV protease cleavage site. The BacMam construct was used to generate baculoviruses for expression of 5-HT3A in HEK293F cells. The proteins were solubilized from the membrane with the detergent C12E 9, and purified using MBP affinity chromatography. The affinity tag was removed by TEV protease treatment and immobilized metal ion affinity chromatography. The receptors were further purified by size-exclusion chromatography (SEC).

SUBMITTER: Wu ZS 

PROVIDER: S-EPMC4564879 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature