Overview of Protein Purification and Characterization

Protein purification has a 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. 

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. 

It is often necessary to purify a particular protein to better understand its role in the nutritional value and physicochemical properties of food. Similarly, many enzymes have been purified to study their effect on the texture, color, flavor, and nutritional value of foods. The purification and characterization of protein-based microbial toxins has been necessary to better understand their mechanisms of action and their roles in food-borne disease. 

Many proteins 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 starting materials, and 

Many publications in the area of protein research are entitled Purification and characterization of. and describe a purification procedure in sufficient detail that it can be reproduced in another laboratory. The characterization section may include structural, functional, and genetic information, and carrying out such studies is likely to require at least milligram quantities of pure protein. Ideally the purification should involve a small number of steps, with good recovery at each step. If the recovery is poor ( 50% at any step), however, there should be some indication of what happened to the missing activity. Has it been discarded in the other fractions for the sake of purity, or does it represent a true loss of activity If the latter, then the end-product may be less than fully active despite apparent homogeneity indicated by standard analysis. The choice between recovery and purification at each step can be problematic taking a narrow cut of a chro- 

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B4.1 Overview of Protein Purification and Characterization B4.2 Overview of Conventional Chromatography... 

The physicochemical and other properties of any newly identified drug must be extensively characterized prior to its entry into clinical trials. As the vast bulk of biopharmaceuticals are proteins, a summary overview of the approach taken to initial characterization of these biomolecules is presented. A prerequisite to such characterization is initial purification of the protein. Purification to homogeneity usually requires a combination of three or more high-resolution chromatographic steps (Chapter 6). The purification protocol is designed carefully, as it usually forms the basis of subsequent pilot- and process-scale purification systems. The purified product is then subjected to a battery of tests that aim to characterize it fully. Moreover, once these characteristics have been defined, they form the basis of many of the QC identity tests routinely performed on the product during its subsequent commercial manufacture. As these identity tests are discussed in detail in Chapter 7, only an abbreviated overview is presented here, in the form of Figure 4.5. 

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