Overview of Protein Purification and Characterization


Protein purification has an over 200-year history: the first attempts at isolating substances from plants having similar properties to “egg albumen,” or egg white, were reported in 1789 by Fourcroy. Many proteins from plants were purified in the nineteenth century, though most would not be considered pure by modern standards. A century later, ovalbumin was the first crystalline protein obtained (by Hofmeister in 1889). The year 1989 may not go down in history as a milestone in protein chemistry, but since then there has been a resurgence of interest in proteins after more than a decade of gene excitement.

protein-purification-Fourcroy protein-purification-Hofmeister

The aims of protein purification, up until the 1940s, were simply academic. To then, even the basic facts of protein structure were not fully appreciated, and pure proteins were needed just to study structure and test the rival theories of the pre-DNA days. During the Second World War, an acute need for blood proteins led to development of the Cohn fractionation procedure for purification of albumin and other proteins from serum (Cohn et al., 1946). This was the inception of large-scale protein purifications for commercial purposes; Cohn fractionation continues to be used to this day.

The nature of the proteins studied has also changed substantially. Whereas enzymes were once the most favored subjects, they have now been superceded by nonenzymatic proteins such as growth factors, hormone receptors, viral antigens, and membrane transporters. Many of these occur in minute amounts in the natural source, and their purification can be a major task. Heroic efforts in the past have used kilogram quantities of rather unpleasant starting materials, such as human organs, and ended up with a few micrograms of pure product. It is now more usual, however, to take the genetic approach: clone the gene before the protein has been isolated or even properly identified, and then express it in a suitable host cell culture or organism. The expression level may be orders of magnitude higher than in the original source, which will make purification a relatively simple task. It can be useful to know beforehand some physical properties of the protein, to facilitate the development of a suitable purification protocol from the recombinant source. On the other hand, there are now several ways of preparing fusion proteins, which can be purified by affinity techniques without any knowledge of the properties of the target protein. Moreover, there are ways of modifying the expressed product to simplify purification further.


For many people embarking on a protein purification project, there is no choice of material. They are studying a particular biological tissue or organism, and the objective is to purify a protein from that source. However, there may be approaches that can make the project simpler. If, for instance, the source is difficult to obtain in large amounts, it may be best to carry out at least preliminary trials on a source species more readily obtained. The most obvious and relevant example is when the species being studied is Homo sapiens, and tissue samples are not readily available for practical or ethical reasons, or both. In this case, it is usual to go to where mammalian tissue is readily available (i.e., an abattoir) and work with bovine, ovine, or porcine sources. Alternatively, if quantity of tissue is not a problem, the humble laboratory rat may suffice. Once a protocol for purifying the protein from substitute sources has been worked out, it will be much easier to develop one using human material—the identical procedure may work satisfactorily. Proteins differ to a fairly small extent between species that have diverged within about 100 million years, a time frame that groups together most higher mammals. Thus the behavior of proteins derived from different animals with respect to the various fractionation procedures is likely to be similar, and a protocol worked out for pig tissues is likely to need only minor adjustments for application to human tissues.


The methods available for protein purification range from simple precipitation procedures used since the nineteenth century to sophisticated chromatographic and affinity techniques that are constantly undergoing development and improvement. Methods can be classified in several alternative ways—perhaps one of the best is based on the properties of the proteins that are being exploited. Thus the methods can be divided into four distinct but interrelated groups depending on protein characteristics: surface features, size and shape, net charge, and bioproperties.

Reference: Current Protocols in Molecular Biology