title: Solubilization of native integral membrane proteins in aqueous buffer by noncovalent chelation with monomethoxy poly(ethylene glycol) (mPEG) polymers.
abstract: Highly hydrophobic integral membrane proteins (IMPs)are typically purified in excess detergent media, often resulting in rapid inactivation and denaturation of the protein. One promising approach to solve this problem is to couple hydrophilic polymers, such as monomethoxypolyethylene glycol (mPEG) to IMPs under mild conditions in place of detergents. However, the broad application of this approach is hampered by poor reaction efficiencies, low tolerance of detergent stabilized membrane proteins to reaction conditions, and a lack of proper site-specific reversible approaches. Here, we have developed a straightforward, efficient, and mild approach to site-specific noncovalent binding of long-chain polymers to recombinant IMPs. This method uses the hexa-histidine tag (His-Tag) often used for purification of recombinant proteins as an attachment site for mPEGs. Solubility studies performed using five different IMPs confirmed that all tested mPEG-bound IMPs were completely soluble and stable in detergent free aqueous buffer compared to their precipitated native proteins under the identical circumstances. Activity assays and circular dichroism (CD) spectroscopy confirmed the structural integrity of modified IMPs.
authors: The Joint Center for Structural Genomics and The Scripps Research Institute, Department of Molecular Biology, La Jolla, California 92037, United States.
authors: Thamara K
authors: TK
authors: Janaratne
authors: Linda
authors: L
authors: Okach
authors: Ansgar
authors: A
authors: Brock
authors: Scott A
authors: SA
authors: Lesley
mesh_terms: D002021:Buffers; D002614:Chelating Agents / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification*; D011092:Polyethylene Glycols / Q000737:chemistry*; D011108:Polymers / Q000737:chemistry*; D055550:Protein Stability / Q000187:drug effects*; D012995:Solubility
chemical_list: D002021:Buffers; D002614:Chelating Agents; D008565:Membrane Proteins; D011108:Polymers; D011092:Polyethylene Glycols; C028210:monomethoxypolyethylene glycol