Membrane protein purification and reconstitution

The first step of the purification of an integral membrane protein is to disrupt its interactions with other integral proteins and the lipids in the membrane.  This is generally achieved through adding a detergent that solubilizes the membrane.  In the order to solubilize membrane but not denature the protein gentle detergents like as octyl glucoside or Triton X-100 is used rather than the harsh detergent SDS.  they readily  intercalate  into  the  lipid  disrupt and bilayer  the  hydrophobic  interactions as  the  detergent  molecules  are  themselves  amphipathic  .

At Once solubilized, the hydrophobic region of the integral protein is coated with a layer of detergent molecules that enables the protein to remain in solution. The solubilized protein can then be purified as for a water-soluble

                                  Figure:  Structures of (a) Triton X-100 and (b) octyl glucoside.

   Figure:  (a) Detergent solubilization and (b) reconstitution into artificial lipid vesicles of an integral membrane protein.

globular  protein  as long  as detergent  is reserved  in the  buffers  to avoid  aggregation  and loss of the protein.  Once the purified, an integral protein can be reincorporated into artificial lipid vesicles liposomes in order to learning its function in above figure. If the phospholipids  are added to the protein in detergent solution  and  detergent  dialyzed  away the  phospholipid   vesicles  holding  the protein  will  spontaneously   form.  The method of the protein can then be studied. For instance, if the Ca²⁺   -ATPase is reincorporated  into lipid vesicles then its function  example transport  of Ca²⁺ upon ATP hydrolysis  can be studied  via monitoring ATP to the outside and  Ca²⁺ on the inside  of the vesicle  upon addition  of Ca²⁺.