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Expression and detergent free purification and reconstitution of the plant plasma membrane Na+/H+ antiporter SOS1 overexpressed in Pichia pastoris.
Biochimica et Biophysica Acta (BBA) - Biomembranes ( IF 3.4 ) Pub Date : 2019-10-31 , DOI: 10.1016/j.bbamem.2019.183111
Debajyoti Dutta 1 , Mansoore Esmaili 1 , Michael Overduin 1 , Larry Fliegel 1
Affiliation  

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.

中文翻译:

在毕赤酵母中过表达的植物质膜Na + / H +反向转运蛋白SOS1的表达和无洗涤剂的纯化和重建。

拟南芥的植物质膜Na + / H +反向转运蛋白SOS1(盐覆盖敏感性1)是将Na +挤出细胞外以换取细胞内H +的主要转运蛋白。钠挤压工艺可维持较低的细胞内Na +浓度,从而有助于提高耐盐性。拟南芥SOS1由1146个氨基酸组成,前450个位于N端膜转运结构域,其余形成胞质调节结构域。为了研究蛋白质的特征,使用表达载体pPICZA,克隆了包含残基28至460和28至990的两种不同的SOS1构建体,并在具有C末端组氨酸标签的毕赤酵母的多甲基酵母菌株中过表达。苯乙烯苹果酸共聚物(SMA)被用作洗涤剂的经济有效替代品,以溶解和分离该膜蛋白。固定化的Ni2 +离子亲和色谱用于纯化表达的蛋白质,每公升巴斯德毕赤酵母培养物的收率约为0.6-2 mg SOS1。将含有28至990位氨基酸的SMA纯化蛋白直接重构为脂质体,以确定Na +转运活性,并且具有功能活性。但是,用氨基酸28-460进行的类似重组不能产生功能蛋白。其他结果表明,在氨基酸481处截短的SOS1蛋白具有活性,这暗示了在残基461-481之间存在SOS1活性所必需的元素。
更新日期:2019-11-01
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