Protein purification tutorial: A common workflow for lab-scale purification


Hi, I am Ana. Today I will talk about a common
workflow for protein purification at lab-scale. Before the chromatographic work starts preparations
are needed: First we need to produce the protein of interest, our target protein, and then
we need to remove gross impurities. Proteins are very complex, and it is not easy
to synthetize them. That is why it is common to utilize microorganisms
to produce proteins. A cell, for example this bacterium, is genetically
modified to utilize the machinery used to produce its own proteins to also make the
protein of interest. The bacteria are then cultivated to generate
more of the protein of interest. Usually, this is done in a culture flask with
cell culture growth medium. We add salt, sugar, proteins, and water to
enable growth of the bacteria. After a day or so, bacteria have had time
to double in numbers several times and they are ready for harvest and wash. To extract the protein, we need to disrupt
the bacteria. Bacterial debris is discarded, and we may
then focus on the protein extract. The protein extract will look like this. Here we have all the water-soluble molecules
that were in the bacteria. Now, the chromatographic purification starts. We will first isolate the target protein from
bacterial host proteins and other molecules. To facilitate purification, a tag has been
introduced to the target protein. The most common tag is an array of histidine
amino acids, referred to as His-tag. This is used because histidine has high affinity
to metals and therefore binds to metal-containing chromatography resins, which are used to purify
tagged proteins. This type of chromatography resin is called
an immobilized metal affinity resin, or IMAC for short. Guess what happens if you attach an array
of histidine molecules to the target protein and then pass the cell extract through an
IMAC resin? The target protein with the histidine tag
will bind to the IMAC resin and the rest will pass through. This type of purification is called affinity
chromatography. Now, how does this work in practice? We pass the cell extract through the resin
and then we wash off impurities. Conditions of the fluid are then changed so
that the tag loses its affinity for the resin and the target protein is released; this is
known as elution of the protein from the resin. After this step, the sample volume is usually
reduced, and the target protein concentrated. This sample is now highly enriched with target
protein. The target protein is probably the most abundant protein in the sample; however, if the purity is not high enough, additional chromatography
steps may be needed. A common second chromatography step is size
exclusion chromatography, SEC. In SEC, proteins are separated according to
their size. The bigger the molecule, the faster it passes
through the column. We can’t see proteins with the naked eye,
which means that we can’t see when the protein of interest elutes (or leaves) the column. However, proteins absorb UV-light, and because
of this, they can be detected with a UV-monitor. The UV monitor of ÄKTA™ systems is connected
to the outlet of the column and will provide information about when the protein elutes. ÄKTA systems synchronize protein UV absorbance
with elution volume and position in the fraction collector. In this example, most of the protein elutes
in fractions F3 and F4. Finally, here we have our protein. Now the interesting part starts as we have
generated the starting material for most of the experiments performed in research laboratories. I wish you all the best with your protein
purification.

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