The solubilisation and purification of an intrinsic membrane protein presents problems distinct from those encountered in purifying a conventional soluble protein. Discuss this statement.

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The solubilisation and purification of an intrinsic membrane protein presents problems distinct from those encountered in purifying a conventional soluble protein. Discuss this statement.

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In order to answer the question, this essay will first describe how soluble proteins are purified. It will then describe the process of solubilising an integral membrane protein specifically, and demonstrate differences between the two processes.

There are several methods for the purification of proteins in aqueous solution. Since these methods discriminate based on one characteristic that may be shared by several proteins, it is almost always necessary to use multiple methods to purify a protein from its cellular environment. First, the cell must be homogenised in order to make all the proteins within available. In theory, this presents a problem since proteins are mixed with proteases, and could be degraded. In practice vacuoles form spontaneously and quickly to mitigate this effect so it is not a problem that has to be contended with. After homogenisation, there are several chromatographic methods available to purify proteins completely. Size exclusion chromatography separates proteins based on molecular weight, as smaller proteins are retarded by the resin and so take longer to flow through. Ion exchange chromatography involves charged resin which binds charged amino acid residues. A buffer containing a high competing ion concentration is used to elute the proteins by binding them, and displacing them from the resin. The required strength of the elution buffer depends on the proteins’ isoelectric points, so by increasing the strength of the buffer, different proteins can be eluted at different times. Affinity chromatography works in a similar, but more specific way. Resin beads are labelled by antibodies which bind specific proteins. The proteins are then eluted by a buffer containing a secondary antibody which competes with the stationary antibody binding the protein, allowing the protein to elute. A high salt concentration can also be used to elute the protein by disrupting the non-covalent forces which attach it to the primary antibody. Hydrophobic interaction chromatography uses resin bound with phenyl or octyl groups to interact and separate proteins based on hydrophobicity. Using the correct method - or combination of methods - is the principal problem that needs to be solved when purifying a water soluble protein.

The important difference between non-membrane proteins and integral membrane proteins is that the latter group are not water soluble. The entire surface of the protein is held in a phospholipid bilayer by entropically driven hydrophobic forces made possible by the fact that the amino acid residues forming the part of surface which is held within the membrane, contain non-polar R-groups. This characteristic of the R-groups is what precludes the protein from being water soluble. All protein purification methods require the protein to be in aqueous solution, so insoluble proteins must be altered to allow this condition. Integral membrane proteins differ from peripheral membrane proteins, which are attached to the outside of the membrane by interactions with a particular phospholipid head group. These can be removed from the membrane relatively easily by disrupting those interactions, usually by means of a solution of high ionic strength1. Once removed from the membrane peripheral proteins are readily soluble since they have a hydrophilic surface. Anchored membrane proteins are also a different classification. They have a membrane spanning helix (usually an alpha-helix) which attaches them to the membrane, but the surface of the protein is still hydrophilic. These can be solubilised by cleaving off the membrane spanning domain with a targeted protease such as trypsin2. Proteolysis cannot be used to solubilise integral membrane proteins, since by definition, removing all membrane spanning regions on such a protein would destroy it.

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Figure 1 – membrane proteins. Shown in green is an anchored membrane protein, in blue is an integral membrane protein and in red a peripheral membrane protein.

In order to remove an integral membrane protein from its environment, detergents must be used. Described broadly, a detergent is a hydrophobic organic molecule with a hydrophilic “head group”. In solution, these molecules aggregate to form micelles, a sphere in which the hydrophobic tails are on the inside while the head groups face outwards into the aqueous solvent. At concentrations high enough to form micelles (the critical micelle concentration (CMC))3, detergents can ...

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