A novel method for expressing and purifying large quantities of functional and stable human voltage-gated proton channel (hHv1).

Abstract

Purifying membrane proteins has been the limiting step for studying their structure and function. The challenges of the process include the low expression levels in heterologous systems and the requirement for their biochemical stabilization in solution. The human voltage-gated proton channel (hH_v1) is a good example of that: the published protocols to express and purify hH_v1 produce low protein quantities at high costs, which is an issue for systematically characterizing its structure and function. Based on a pipeline approach, we developed a novel method to produce large quantities of properly folded and fully functional hH_v1. We found that using the correct Escherichia coli strain in an autoinduction medium at low temperatures maximized protein expression. Furthermore, solubilization screenings showed that the detergent Anzergent 3-12 was a better alternative than Fos-choline-12 to purify hH_v1, considerably reducing the costs. Buffers with high ionic strength increased the protein extracted during detergent solubilization and the stability of hH_v1 during downstream processing. Finally, a further improvement was achieved when an enterokinase cutting site was inserted at the N-terminus of the protein. Our novel method produces properly folded and fully functional hH_v1, increasing the protein yield by 100 times and reducing the cost by 96% while improving the protein stability compared to the previously published protocols. Our work will accelerate studies on hH_v1 and its possible future therapeutic use, while serving as an example for developing purification methodologies for other challenging membrane proteins.