Expression and Purification of Mammalian Bestrophin Ion Channels

Summary

The purification of ion channels is often challenging, but once achieved, it can potentially allow in vitro investigations of the functions and structures of the channels. Here, we describe the stepwise procedures for the expression and purification of mammalian bestrophin proteins, a family of Ca²⁺-activated Cl^- channels.

Abstract

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca²⁺-activated Cl^- channel (CaCC) predominantly expressed in retinal pigment epithelium (RPE). The physiological and pathological significance of BEST1 is highlighted by the fact that over 200 distinct mutations in the BEST1 gene have been genetically linked to a spectrum of at least five retinal degenerative disorders, such as Best vitelliform macular dystrophy (Best disease). Therefore, understanding the biophysics of bestrophin channels at the single-molecule level holds tremendous significance. However, obtaining purified mammalian ion channels is often a challenging task. Here, we report a protocol for the expression of mammalian bestrophin proteins with the BacMam baculovirus gene transfer system and their purification by affinity and size-exclusion chromatography. The purified proteins have the potential to be utilized in subsequent functional and structural analyses, such as electrophysiological recording in lipid bilayers and crystallography. Importantly, this pipeline can be adapted to study the functions and structures of other ion channels.

Introduction

Bestrophins are a family of ion channels conserved through species varying from bacteria to humans¹. In humans, the BEST1 gene, located on chromosome 11q12.3, encodes the membrane protein Bestrophin-1 (BEST1) that is predominantly expressed in the basolateral membrane of retinal pigment epithelium (RPE) cells of the eyes^2,3,4. Comprised of 585 amino acids, the first ~350 of which are highly conserved among species and contain its transmembrane region, BEST1 acts as a CaCC in humans^1,5<....

Protocol

1. Producing BacMam Expression Baculoviruses

1. Insert the coding sequence of a desired mammalian bestrophin protein into the pEG BacMam vector¹⁸ with a Tobacco Etch Virus (TEV) protease recognition sequence, followed by a GFP-10x His-tag at the C-terminus of the protein.
2. Transiently transfect the expression plasmid into adhesive HEK293 cells^{19,20,21,22,23}. Check the expression of the GFP-fusion protein under a fluorescent microscope with 10X or 20X magnification, ....

Results

The fluorescence intensity in transiently-transfected adhesive HEK293 cells (Figure 1A) is a good indicator for the projected protein expression level in suspension HEK293-F cells (Figure 1B). If the target protein is not well-expressed or is mis-localized in HEK293 cells after transient transfection, it is recommended to consider modifying the expression construct (e.g., changing the position of the GFP tag or making mu.......

Discussion

This protocol describes a useful pipeline for expression and purification of mammalian bestrophin ion channels to be used for future in vitro analyses. While the FPLC device is required for size-exclusion chromatography, a syringe pump is sufficient for all steps of affinity chromatography including binding, washing, and eluting. When using a syringe pump to push solutions (in a syringe) through a column, it is essential to underlay the spring side to avoid pushing air bubbles into the column. If the purity of t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
HEPES	Fisher Scientific	AC327265000
NaCl	Fisher Scientific	AC446212500
Glycerol	Fisher Scientific	G33-500
Imidazole	Fisher Scientific	AC301870010
MgCl2	Fisher Scientific	AC197530010
TCEP	Fisher Scientific	AA4058704
Aprotinin	Fisher Scientific	AAJ63039MA
Leupeptin	Fisher Scientific	AAJ61188MB
Pepstatin A	Fisher Scientific	AAJ20037MB
Phenylmethylsulfonyl fluoride	Fisher Scientific	AC215740050
DDM sol-grade	Anatrace	D310S
DDM anagrade	Anatrace	D310
Sf-900 II SFM	ThermoFisher	10902179
FreeStyle medium	ThermoFisher	12338018
NanoDrop spectrophotometer	ThermoFisher	ND-2000
High pressure homogenizer	Avestin	Emulsiflex-C5
HisTrap column	GE	17-5248-01
Superdex-200 column	GE	28990944
AKTA Pure	GE	29018224
Ultra-15 centrifugal filter units	Millipore	UFC910024
Ultra-4 centrifugal filter units	Millipore	UFC810024
Ultra-0.5 centrifugal filter units	Millipore	UFC505024
Optima XE-90 Ultracentrifuge	Beckman Coulter	A94471
Mini-PROTEAN Tetra Cell	Bio-Rad	1658004
Mini-PROTEAN precast gel	Bio-Rad	4561084
T100 Thermal Cycler	Bio-Rad	1861096
PolyJet transfection reagent	SignaGen	SL100688
pEG BacMam vector			Obtained from the Gouaux lab at Vollum Institute