“Affinity chromatography” describes a range of chromatographic techniques in which the beads in the column have some sort of affinity for the protein of interest (POI). In the hand-drawn introductory video, you saw how lactose bound to a bead can be used to purify lactase. Lactose is a sugar; lactase is an enzyme that binds lactose. More technically, lactose is a ligand for lactase. Ligand describes a molecule that binds to another (usually larger) molecule.
Some affinity chromatography techniques rely on antibodies. Antibodies are proteins made by our immune system that bind tightly to foreign molecules, targeting them for destruction. (Vaccines work by giving our immune system a safe exposure to a pathogen, so that when the real pathogen arrives, we already have the ability to make antibodies against it and can fight it off quickly.) It is possible to make antibodies that bind tightly to most POIs. By binding the antibodies to beads, you can capture the POI.
In the final project, you’ll do metal affinity chromatography. In BTEC 1100, you cloned the gene BglA. The plasmid you cloned BglA into was engineered to add a his tag to the BglA coding sequence; this means that, at the end of the BglA protein, there are extra six histidine residues added. Six histidine residues in a row tend to bind to the metal nickel. In a sense, the his tag provides a ”handle” that allows us to “grab” (bind to beads) whatever protein we attach the handle to. His tags, and other tags that can serve as “handles”, are often added to proteins to make them easier to purify.
The method of elution varies depending on the type of affinity being used. Common methods of elution include altering the pH or increasing the salt concentration; both of these changes can disrupt ionic and hydrogen bonding that may be binding the proteins to the beads. In the metal affinity chromatography you’ll use to purify BglA, you will use a competitive ligand. While the nickel on the beads likes to bind to the his tag, it likes to bind to the molecule imidazole even more. By slowly increasing the concentration of imidazole, you will elute the his-tagged POI from the nickel column.
The screenshot below shows an elution profile from an affinity chromatography experiment. The line that starts out grey shows the protein coming off the column; the red line indicates the changing composition of the buffer that promotes elution. Watch the video to see what’s happening in the column to generate that elution profile.
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