Expression and Purification of the Human Voltage-Gated Proton Channel (hHv1).

Abstract

The voltage-gated proton channel (H_v1) is a membrane protein that dissipates acute cell proton accumulations. To understand the molecular mechanisms explaining H_v1 function, methods for purifying the protein are needed. Previously, methods were developed for expressing and purifying human H_v1 (hH_v1) in yeast and later in bacteria. However, these methodologies produced low protein yields and had high production costs, considerably limiting their usefulness. The protocol described in this work was developed to overcome those limitations. hH_v1 is overexpressed in bacteria, solubilized with the detergent Anzergent 3-12, and purified by immobilized metal affinity chromatography (IMAC) and size-exclusion chromatography (SEC). Our protocol produced higher protein yields at lower costs than previously published methodologies. Key features • hH_v1, containing a poly-His tag followed by an enterokinase cutting site in its N-terminus, is overexpressed in E. coli by autoinduction. • The detergent Anzergent 3-12 is used to solubilize and purify hH_v1 using nickel-immobilized metal affinity chromatography (IMAC). • The entire procedure can be performed in 6 days.