Protein Purification

Protein Purification


Proteins are life’s way of getting things
done. From structural support to catalyzing reactions,
proteins have numerous functions, depending on the physiological need. In order to gain a better understanding of
these proteins and their functions, we want to know that we’re actually working with the
protein we want without the presence of other impurities. Based on knowledge about different proteins
and their properties, and with a little help from technology, scientists and researchers
have developed methods to separate target proteins from complex mixtures. This is known as “Protein Purification.” Protein purification is a fundamental concept
for aspiring biochemists. Isolated proteins are always being used. From catalyzing important reactions for pharmaceuticals,
to understanding the different functions of proteins through research, to cleaning up
environmental messes, the applications for pure proteins are nearly endless. in our experiment for Protein Purification,
we wanted to isolate Alkaline Phosphatase, or AP, from E. Coli cells, specifically, the
K-12 strain. AP, along with many other proteins, are found
within the periplasm of E. Coli cells, located between the outer membrane and plasma membrane. This is the starting point of the experiment
and where we will base our first purification technique. Since most proteins are found inside of cells,
we want to use a method that would release our protein of interest into a soluble solution
while reducing the number of unwanted particulates. In our case, we created an Osmotic Shock solution
to disrupt only the outer membrane of E. coli to release the contents of the periplasm,
which contains AP and other proteins. From there, What’s left in the mixture are
the proteins from the periplasm, including AP, and the remains of the E. Coli cell. To group up the cell debris, we centrifuged
the mixture at 10,000 rpm to move it all down to the bottom of the tube. This solid mass at the bottom is called the
“pellet,” and the remaining liquid is the “supernatant,” which contains AP. We want to collect this mixture for the next
purification steps After this step, we have exposed AP to the
mixture and removed the E. Coli cells, but this is only a “fraction” of the process. For our second purification step, we take
advantage of a protein’s tendency to denature and unfold when heated up to high temperatures,
by breaking the intermolecular forces that hold it together. Magnesium Sulfate is added to protect AP from
denaturing, leaving most of the unwanted proteins to precipitate out of the solution. Similar to the previous purification step,
we centrifuged all of the precipitate down to the bottom, discarded the unwanted pellet,
and collected the supernatant containing our protein of interest, further purifying our
mixture. Moving on to the next step, we want to change
the composition of the solution for the final purification step while retaining our protein. Normally, our protein is surrounded by a solvent
shell that allows it to be soluble in the solution. Saturating the solution with a salt, like
Ammonium Sulfate, dehydrates our protein making it insoluble, which precipitates it out of
the solution. Just like before, the solution is centrifuged
to move all of the precipitate to the bottom of the tube. Except this time, the pellet contains our
protein and is less dense than the solution, causing it to float to the top after centrifuging
was completed. We carefully pipetted out the unwanted supernatant
and dissolved the pellet in dialysis buffer to prepare for the dialysis. The dissolved protein mixture was then transferred
into a dialysis tube, which was subsequently suspended in a beaker full of dialysis buffer. This was allowed to run for a couple of hours
to fully equilibrate the inside of the dialysis tube with the external dialysis buffer. Let’s zoom in to see how the semi permeable
dialysis membrane works. The membrane contains several specifically
sized pores ranging from 3 – 30 kDa depending on the size of the molecules in the solution. In this case, the pores allow ammonium sulfate
to diffuse out of the dialysis tube while being displaced by buffer ions. Our large protein is too big to fit through
the pores and remains in the dialysis tube. This process can be repeated multiple times
to further clean the solution. After the dialysis is complete, we transferred
the contents of the dialysis bag into microfuge tubes to centrifuge to remove any particulate
material and unwanted protein. After discarding the pellet, the supernatant
was then filtered through a syringe to further cleanse the solution. At this point, we have successfully changed
the composition of the mixture and are ready to use it and load it into the FPLC. Now, with the assistance of technological
machinery, we can really isolate AP from any remaining impurities in the solution. The protein solution is run through FPLC,
which stands for Fast Protein Liquid Chromatography. We used the AKTA Start by GE which featured
an anion exchange column, conductivity reader, and selective pump. The pump is able to move the protein through
a mobile phase with varying concentrations of NaCl salt buffer to run through the Anion
Exchange Column. The Anion Exchange Column is a form of liquid
chromatography where the column is filled with beads that hold positively charged ions
to bond with negatively charged molecules moving through the column. Negative ions in the mobile phase, including
our protein, AP, will bind to these beads. As the conductivity increases, Cl- ions “wash”
off our protein into our fractions. When we plot the conductivity and absorbance,
we can see that the conductivity increased over the run, and there is a huge spike of
absorbance under one of the fractions. The fraction at this conductivity contains
our AP! And after all of our hard work, we have purified
Alkaline Phosphatase from E. Coli! Overall, we should see a decrease in total
protein and a lesser or equal amount of protein activity after each purification step. The small decrease in activity is due to the
inevitable loss of our protein when performing a purification of any sort. Generally, a protein assay, like the Bradford
assay used in our experiment, would be performed to quantify total protein, while the kinetic
assay would be used to measure the enzyme activity. Together, we can use this data to assess our
protein purity. In addition to this, we can also measure our
protein purity through SDS-PAGE. We can visualize our gel using methods like
silver staining and western blotting/immunoblotting. Based off of the standards, these methods
gives us the molecular weights of the proteins in each purification step. As the mixture becomes more pure, we should
see higher quality bands of our protein. Thanks for watching! I hope you all enjoyed our video and gained
a better understanding on some of the methods of protein purification! Be sure to comment the video, like the video,
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