Chemical Engineering, School for Engineering of Matter, Transport and Energy, Arizona State University, Tempe, AZ, 85287, USA; Center for Applied Structural Discovery, The Biodesign Institute, Arizona State University, Tempe, AZ, 85287, USA.
Chemical Engineering, School for Engineering of Matter, Transport and Energy, Arizona State University, Tempe, AZ, 85287, USA.
Protein Expr Purif. 2020 Nov;175:105716. doi: 10.1016/j.pep.2020.105716. Epub 2020 Jul 29.
The high-flux/low-affinity cyanobacterial bicarbonate transporter BicA is a member of sulfate permease/solute carrier 26 (SulP/SLC26) family and plays a major role in cyanobacterial inorganic carbon uptake. In order to study this important membrane protein, robust platforms for over-expression and protocols for purification are required. In this work we have optimized the expression and purification of BicA from strain Synechocystis sp. PCC 6803 (BicA) in Escherichia coli. It was determined that expression with C43 (DE3) Rosetta2 at 37 °C produced the highest levels of over-expressed BicA relative to other strains screened, and membrane solubilization with n-dodecyl-β-d-maltopyranoside facilitated the purification of high levels of stable and homogenous BicA. Using these expression and purification strategies, the final yields of purified BicA were 6.5 ± 1.0 mg per liter of culture.
高流量/低亲和力蓝细菌碳酸氢盐转运蛋白 BicA 是硫酸盐转运体/溶质载体 26（SulP/SLC26）家族的成员，在蓝细菌无机碳吸收中起主要作用。为了研究这种重要的膜蛋白，需要有可靠的过表达平台和纯化方案。在这项工作中，我们优化了来自集胞藻 PCC 6803（BicA）的 BicA 在大肠杆菌中的表达和纯化。结果表明，与筛选的其他菌株相比，在 37°C 下用 C43（DE3）Rosetta2 表达可产生最高水平的过表达 BicA，并用正十二烷基-β-D-麦芽糖苷进行膜溶解有助于纯化出高浓度稳定均一的 BicA。使用这些表达和纯化策略，最终每升培养物可获得 6.5±1.0mg 的纯化 BicA。
