Expression and detergent free purification and reconstitution of the plant plasma membrane Na+/H+ antiporter SOS1 overexpressed in Pichia pastoris

Abstract

The plant plasma membrane Na⁺/H⁺ antiporter SOS1 (Salt Overlay Sensitive 1) of Arabidopsis thaliana is the major transporter extruding Na⁺ out of cells in exchange for an intracellular H⁺. The sodium extrusion process maintains a low intracellular Na⁺ concentration and thereby facilitates salt tolerance. A. thaliana SOS1 consists of 1146 amino acids, with the first 450 in a N-terminal membrane transport domain and the balance forming a cytosolic regulatory domain. For studies on characterization of the protein, two different constructs of SOS1 comprising of the residues 28 to 460 and 28 to 990 were cloned and overexpressed in methylotropic yeast strain of Pichia pastoris with a C-terminal histidine tag using the expression vector pPICZA. Styrene malic acid copolymers (SMA) were used as a cost-effective alternative to detergent for solubilization and isolation of this membrane protein. Immobilized Ni²⁺-ion affinity chromatography was used to purify the expressed protein resulting in a yield of ~0.6-2 mg of SOS1 per liter of Pichia pastoris culture. The SMA purified protein containing amino acids 28 to 990 was directly reconstituted into liposomes for determination of Na⁺ transport activity and was functionally active. However, similar reconstitution with amino acids 28-460 did not yield a functional protein. Other results have shown that the truncated SOS1 protein at amino acid 481 is active, which infers the presence of an element between residues 461-481 which is necessary for SOS1 activity. This region contains several conserved segments that may be important in SOS1 structure and function.

Keywords: Arabidopsis thaliana; Liposome reconstitution; Membrane protein; Na(+)/H(+) antiporter; Protein purification; Pyranine; SOS1; Salt tolerance.