Production of functional human potassium channel protein KV1.5 in an Escherichia coli-based cell-free protein synthesis system.

Abstract

K_V1.5 is a member of the voltage-gated potassium ion channel family, which plays important roles in cell excitability, ion homeostasis and cell signaling. It is very difficult to rapidly obtain functional ion channel proteins in large quantities. Here, we report the utilisation of an Escherichia coli cell-free protein expression system to efficiently express K_V1.5 in a soluble form over a 16 h period, achieving a yield of up to 1 mg·mL^-1. The synthesised K_V1.5 was purified using a HisTrap HP column to capture the 6 × his tags in the presence of detergent FC-14. The resulting K_V1.5 exhibited α-helical secondary structures as assayed by circular dichroism spectroscopy, and demonstrated binding affinity for its agonist benzocaine through microscale thermophoresis measurements. Both, excitatory and inhibitory effects of benzocaine and vernakalant on reconstituted K_V1.5-liposomes was measured using H⁺-dependent quenching of a fluorescent dye, 9-amino-6-chloro-2-methoxyacridine, to monitor the flux of K⁺. Overall, our findings demonstrated that the functional human ion channel K_V1.5 could be heterologously synthesised via an E. coli-based cell-free protein expression system, which will expand the application scope of the cell-free protein expression system for structure and drug high-throughput screening within the entire voltage-gated potassium channel family.