Protein chromatography - types, tips, & example workflow for protein purification

The basic idea with this is that you can flow a solution containing the protein through columns filled with little beads (resin) and the proteins will get separated based on how they interact with the resin on their journey. Different proteins have different properties, so they will travel faster or slower (in SEC) or “stick” or “not stick” (in affinity chromatography). You can use different resins to separate based on different properties, and you can flow different buffers (pH-stabilized salt waters) through the column to selectively unstick stuck ones based on how stuck they are. http://bit.ly/proteincleaning

I start with an AFFINITY CHROMATOGRAPHY step. This uses resin that recognizes something really specific - usually an affinity tag we’ve added onto the end of the protein (by putting the genetic instructions for it before or after the instructions for our gene in the plasm).

A common affinity tag is a His tag, which is just 6 or 8 Histidines (a specific amino acid), which will bind to a Nickel (Ni) or Cobalt (Co) coated column (Immobilized Metal Affinity Chromatography; IMAC). Other proteins have Histidines too. But not that many in a row, so your protein will bind preferentially. It’ll hog the column and the other proteins will flow through. http://bit.ly/histidineimac

Then you need something that will outcompete the His tag to get your protein off. Bring on the imidazole. It looks like His, so you can flood the column with imidazole to push the His-tagged proteins off. But before you flood it, you wash it with low levels of imidazole to remove non-specific binders that are just binding cuz they happen to have a lot of Hises.

His-tags work well for bacterially-expressed proteins, but the proteins I’m purifying today were expressed in insect cells. Insect cells and cells from other eukaryotes (things with membrane-bound rooms inside their cells to house their DNA, etc. - so basically most stuff except bacteria) have a lot of proteins that naturally have a lot of Hises. So instead of the His tag I’m using a “Strep-tag.” more here: http://bit.ly/streptag

But basically it mimics biotin & binds to a column that mimics streptavadin - the biotin/streptavadin interaction is one of the strongest non covalent (non-electron-sharing) interactions known so these mimics are weakened a little so the protein doesn’t get stuck to the column forever & we can elute it off with another biotin mimic, desthiobiotin. Bacteria naturally have a lot of biotinylated stuff so strep tags don’t work as well for them, but this isn’t as much of a problem with insect cells

Once you’ve eluted your protein (gotten it to come off the column) you have the option to cut off that tag using an endoprotease (protein scissors) that recognizes a sequence between the tag & the start of the protein. After you’ve removed the tag there’s nothing “artificially super specific” about your protein, so you now have to exploit natural differences between your protein and any remaining contaminating proteins (which now includes that protease you added).

The first other property we’ll exploit is charge. Proteins have different charges because they’re made up of different combinations of amino acid letters, some of which are charged.

We’ll take advantage of this using ION EXCHANGE CHROMATOGRAPHY (IEX), where we bind proteins to resins that are oppositely-charged. Ions are charged things and basically you “exchange” ions from salts (like the Na⁺ or Cl⁻ of NaCl (table salt) with protein ions. Then you can gradually increase the salt concentrations so that those salt ions outcompete the protein and you get another exchange. Or you can change the pH to change the protein’s overall charge (the lower the pH, the more free H⁺ for the protein to latch onto & become more positive & vice versa) http://bit.ly/ionexchangechromatography

In CATION EXCHANGE chromatography you have negatively charged resin & you’re binding & exchanging positively-charged (cationic) proteins & salt ions.

ANION EXCHANGE chromatography is the opposite - you have positively charged resin & you’re binding & exchanging negatively-charged (anionic) proteins & salt ions.

Proteins can have similar charges, so there are likely still small levels of lingerers. They might have similar charges but chances are (hopefully) they’ll have different sizes. So next we can use SIZE EXCLUSION CHROMATOGRAPHY (SEC)(aka GEL FILTRATION) to separate the remaining proteins by their size. In this type of chromatography the resin’s “boring” to the proteins so the proteins don’t interact with it. But the resin’s also “bored” in the sense that it has “secret tunnels” “bored” into it. The tunnels have different diameters so proteins have to be small enough to fit in order to go through them. So the smaller the protein, the more tunnels it will go through and the longer it will take to go through the column. So proteins get separated by size, with bigger things coming out sooner, smaller things later.