The Protein Source. Assays

How a particular purification is designed and carried out is very dependent on the source of the protein. It used to be that the only source was from an animal or plant tissue or cultures of cells or microorganisms.

More than likely, the protein of interest was not present at levels any higher than many other proteins and often less, thus requiring a significant enrichment along the way. With over-expressed proteins in recombinant systems, the protein of interest is already enriched since it is usually present at levels much higher than any other protein.

One potential drawback from over-expression of a protein from one species in another species, such as a protein from a eukaryote expressed in bacteria, is the formation of inclusion bodies. These are inactive, insoluble aggregates of protein formed when the expressed protein finds itself in a foreign environment at a very high concentration. In addition, mechanisms for such things as glycosylation, membrane transport, and specific protein folding are not generally found in prokaryotes so that the resultant protein may take up a nonnative conformation.

One potential advantage of a protein forming inclusion bodies is that, if it can be reactivated from the aggregated form, the inclusion bodies themselves can serve as a form of purification (Palmer and Winfield, 2004; Winfield et al., 2004; Singh and Panda, 2005).

The formation of inclusion bodies may also be temperature sensitive. In many instances, failure to obtain soluble protein after expression in Escherichia coli at 37 °C, for example, can be at least partially overcome by expressing the protein at lower temperatures, such as 16 °C. While substantial amounts of protein may still be found in the insoluble pellet, the supernatant may often contain sufficient amounts of active protein.

Obviously, one of the biggest purification steps one can make is to start with an organ or other tissue rather than the whole organism. But, which tissue to use can be just as important since enzymes can be differentially expressed depending on the tissue. For instance, a particular enzyme may be present in high amounts in the kidney but not in the liver.

Therefore, although one may be able to isolate it from the liver, isolating it from the kidney may be easier because there is a lot more to start with and yield will be less of a concern. However, tissues can express specific isoforms differentially so this also should be taken into account when choosing a source.

Assays. Equally important as a good source of protein is a good assay for the protein of interest. It is usually not possible to purify a protein if you cannot identify or locate it as you proceed to separate it from other material. If it does not have enzymatic activity or there is no enzymatic assay available, it could be followed by western blotting or perhaps even simply by following a particular band on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel.

In the case of western blotting, it is possible to make antibodies to a protein without having it in pure form first if the sequence is know from genome studies. This is done by making antibodies to synthetic peptides of hydrophilic stretches of amino acid sequence found in the target protein (Grant, 2002). But remember, you usually isolate what you assay for. If, for instance, your assay detects more than one protein because of cross reactivity, your end product may be different than intended or possibly a mixture of things.

As a protein purification proceeds, one is also interested in determining the total of all protein present in a fraction as a way to judge the efficacy of each step. There are many ways to assay for the total amount of protein, from something as simple as reading the absorbance of a solution (Grimsley and Pace, 2003) to chemical-based colorimetric assays (Olson and Markwell, 2007) and even quantitative amino acid analysis (Rutherford and Dunn, 2011).