In earlier posts I've talked about how to replicate and amplify DNA sequences so that you can make shitloads of proteins. Well, I've realised that I haven't actually spoken about how to extract and purify all of those proteins that you just made (or bullied a cell into making for you).
A lot of these purification processes actually require that you've made a purifiable protein to begin with. For example, with a His tag, you need to add some DNA coding for a bunch of histidine residues to the beginning or end of the sequence. I've actually spoken about His tags in an earlier post: Introduction to Cloning. Essentially the proteins are washed down a column, and the ones with His tags bind to nickel beads in the column. Eventually all that's left in the column are the His-tagged proteins bound to metal beads. These can then be eluted from the column by lowering the pH or by adding extra histidine or imidazole.
A second type of purification process is called GST-Fusion, which of course has absolutely nothing to do with Goods and Services Taxes. GST stands for glutathione-S-transferase. DNA coding for glutathione-S-transferase is positioned right before the DNA for the protein of interest, so that the protein is attached to glutathione-S-transferase. Glutathione-S-transferase attaches to glutathione (a.k.a. GSH), rather than nickel beads. It can be eluted with free glutathione. The GST part can then be removed by cleavage with Xa protease at a site which, like the GST enzyme itself, has also been engineered into the protein.
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