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Membrane protein enrichment
Membrane protein enrichment is the process of increasing the concentration of a native or recombinantly expressed membrane protein in purified cell extracts. Membrane proteins are challenging to purify because they are not water soluble, their expression level is low, they form aggregates when isolated, and they degrade easily. Membrane enrichment begins with the general protein purification steps of: 1) extraction of proteins through cell lysis, 2) precipitation of all proteins in a salt buffer, 3) collection of the precipitated proteins through ultracentrifugation, 4) purification of the desired protein, 5) removal of residual salts, and 6) refolding, if the proteins are denatured (unfolded) during purification. To achieve enrichment, lysis may be performed in several steps to fractionate the cells into membrane, cytoplasmic, nuclear, and cytoskeletal fractions. Often the membranes are then further purified through additional fractionation steps. After isolation of the membranes, high concentrations of detergent are usually added to solubilize the membrane proteins. A high salt buffer to disrupt intramembrane electrostatic interactions can be added as well. After lysis and precipitation, common methods for purification include size-exclusion chromatography, high performance liquid chromatography (HPLC), and affinity purification. When enriching for membrane proteins important considerations are the detergent for solubilization, the purification method for the cell membranes, the ability to refold the protein, and the level of enrichment desired. When expressing the desired membrane proteins as recombinant proteins, inclusion bodies (cytoplasmic aggregates of protein in bacteria) is often helpful to increase the amount of protein available for purification. If maintaining the structure of a membrane protein throughout purification is necessary, techniques that involve the expression of the proteins in the outer membrane of E. coli are available. Membrane protein enrichment is used for structural studies, antibody generation, and drug development. (Credit: Brooke Anderson-White)
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