Optimizing Conditions for the Isolation of Membrane Proteins by Styrene-Maleic Acid Copolymers: A Case Study on E. Coli Inner Membranes

Abstract

Styrene-Maleic acid (SMA) copolymers have emerged as a powerful alternative to detergents for the extraction of membrane proteins from cellular membranes. This approach has been successfully demonstrated for proteins present in the membranes of a variety of organisms including all major systems used for heterologous expression. However, for downstream characterization often high amounts of proteins are required, which remains a challenge in some cases. Extraction of proteins from the membrane and purification of the solubilized material are two major bottlenecks in this process.Here, we report a systematic approach to optimize conditions for the use of SMA to extract the tetrameric potassium channel KcsA from the inner membrane of Escherichia coli, by varying different parameters such as SMA-to-membrane ratio, ionic strength, pH, polymer type and polymer length. Furthermore, conditions for the purification of the channel via Ni-affinity of its poly-His tag have been optimized. Implications of our findings will be discussed in the general context of membrane protein solubilization by SMA.