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Fundamentals of protein chromatography: basic protein purification
Published on July 30, 2020 48 min
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Hello, this is Richard Burgess. I'm a Professor Emeritus of Oncology in the University of Wisconsin-Madison. I'll be speaking to you about the fundamentals of protein chromatography, and in this first lecture on part 1, the basics of protein purification.
In protein purification, the object is to separate a particular protein from all the other proteins and cell components. In E. coli, there are over 4,000 genes and in eukaryotes over 20,000 genes. A given protein may be very non-abundant or it can be very abundant and that will affect the amount of purification that is going to be needed. The goal is not only to separate your protein from other proteins, but from nucleic acids, from carbohydrates, from lipids and from small molecules in the cell. Enzymes, they can be soluble, some are insoluble, some are bound to membranes, some are bound to DNA, they can be present in organelles, in the cytoplasm, in the periplasm, and in the nucleus region. They can be secreted, they can be post-translationally modified, they can be processed.
The goal is to separate one protein from all the rest of the components in the cell. Now, a way to illustrate this is to look at something called the two-dimensional gel of E. coli cell extract. The first dimension is what's called isoelectric focusing, and you separate within a polyacrylamide gel. You create a pH gradient that goes from about pH 4 to pH 10, and then proteins within the gel when subject to an electric field, they will move until they have no charge, that is, their isoelectric point. Proteins vary in their isoelectric points from about 4.5 to over 10. Once you've separated on the basis of isoelectric point, you then lay that piece of gel onto a slab gel and run them on what's called an SDS gel, which is a kind of an electrophoresis that separates on the basis of polypeptide size. The size can vary from on the order of 160,000 in E. coli down to about 10,000. When you do that and then run the gel and then stain it and then de-stain it, you can visualize all the different proteins in the cell. Notice that most of the proteins are in the region between about 160 and 10,000 and between about 4.8 and about 10 isoelectric point. Suppose there is a protein that has an isoelectric point of 6 and a molecular weight of 40 is one of those little spots in the middle of everything. The goal is to separate that from all the others. Now if you were to very high resolution separate and get all the things that had exactly that isoelectric point, and then on it's second step of a purification based on size, get all the things that had a molecular weight of 40 kilodaltons, your protein would be largely pure, but very rarely can one do such a clean separation. One has to use a series of fractionation steps to successively remove components of the cell and other proteins. You may note that in the lower left-hand corner of the 2D gel are some large spots that are very basic. They have isoelectric point of nine or 10 and are very small between 10 and 15,000 daltons. These are ribosomal proteins, very abundant, very small, very basic and most of the proteins you can see are clustered in a slightly more acidic region of the 2D gel.