Purification of proteins using hydrophobic interaction chromatography.

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Ansu Thomas

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Dr Philip Bonner

     Purification of proteins using hydrophobic interaction chromatography.

The emergence of chromatography was in 1903, discovered by a Russian botanist M.S Twsett by separating green leaf pigment using powdered calcium carbonate into a chain of colour coded bands and allowing the solvent to execute through the column packed phase. The name chromatography originates from Greek for color (chromo) and write (graphein). Poole (2003) and Bonner (2007). According to Poole (2003), chromatography is defined as “a physical method of separation and the components are distributed between stationary phase and mobile phase moving in a definite direction.”  The mobile phase is either in liquid or gas state which is flown over the stationary phase, which is a solid medium chosen to separate the residues. Bio molecules like proteins are purified in a similar way, in which the components are introduced to the mobile phase and they are transferred to the column packed stationary phase for separation.  This is mainly due to the protein molecules that have a greater attraction for the stationary phase displaces slowly than the protein molecules that are attracted to the mobile phase. Scopes (1994).

  Different chromatography techniques are used for the purification of proteins; however it determines the sequence of the amino acids. Biotech Articles a(2010). However, Bonner (2007) explains a range of techniques required for protein purification and they are separated according to the difference in their properties GE healthcare (2010). The popular techniques include ion- exchange chromatography which separates ions and charged molecules, affinity chromatography which focuses on the separating proteins by their binding specificities, size- exclusion chromatography separating proteins according to their molecular mass, hydrophobic interaction chromatography and reversed phase chromatography exploiting on the hydrophobic properties of the proteins. (Poole 2003; Bonner 2007; Lehninger 2005). The aim of this essay is to provide different aspects of protein purification using hydrophobic resins.

 Proteins have different sequence of amino acids present in them and their structures include primary, secondary and tertiary. Proteins are purified by hydrophobic interaction chromatography that consists of amino acids with aliphatic functional groups. Majority of these hydrophobic residues are buried inside the three- dimensional protein structure to avoid contact with water, Bonner (2007) where as some of the residues form as hydrophobic patches on the surface of the tertiary structure of protein (figure 1) and they are masked by layers of water molecules to avoid contact with water. However, with the addition of salt, the hydrophobic groups are exposed and this cause to absorb the ordered water layers therefore the interactions between the hydrophobic groups takes place. Wilson and Walker (2005). The solubility of the proteins can be altered by the differences in their ionic strength, temperature and interactions with other hydrophobic regions. GE healthcare (2010)  

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Figure 1:- Tertiary structure of the protein cytochrome C. Yellow indicates the hydrophobic patches on the protein surface and the red indicates the hydrophilic residues.

Image from (GE Healthcare 2010)

It is generally known that proteins are separated by their differences according to charge, size or molecular weight. This difference is exploited by a common procedure called salt fractionation, which adopts ammonium sulphate precipitation. Wilson and Walker (2005); Jason and Osterlund (1997). The concentration of the ammonium sulphate is therefore increased in the protein solution causing the proteins to precipitate and this is due ...

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