The Art and Science of Protein Purification

Medicilon’s protein scientists have been working on protein expression and purification for many years. We can start your project even you have nothing in hand but the name of your protein. In Medicilon’s laboratories, protein purification is performed in scales from micrograms and milligrams. All Protein Purification Services start with the analysis of physico-chemical and biological properties of a target protein resulting in the development of tailored procedures for its extraction, purification and characterization. Email: marketing@medicilon.com.cn web: www.medicilon.com

Obtaining protein In any experiment concerning a protein, it is obviously necessary to obtain material to work with. Most proteins are used by several species. Some proteins are produced only in specific places within an organism, or only at certain times during the lifetime of the organism. The first step is choosing a source for the protein, and the species from which to isolate it. One option, which was historically the only option, and is still a common method, is to find a natural source. This means choosing both the organism of choice and organ with the highest-level expression of the protein of choice. Ideally, you want to find a source that is readily available and reasonably inexpensive. In many cases, issues of cost or availability of source material may govern the source species or even the protein of interest. As an example, hemoglobin was heavily studied during the early years of biochemistry because it is both present at very high concentrations in blood, and was easily isolated from blood samples. Natural sources tend to have problems. The most obvious problems are that many natural sources are difficult to obtain, or may be from less than ideal species, or may produce only very small amounts of the protein of interest. Obtaining a protein from a natural source is especially difficult for human proteins. This is one reason that studies in experimental animals are so popular: it is possible to obtain rabbit livers or cow livers in reasonably large amounts, while obtaining normal human liver is far more difficult. Natural sources do have the advantage that the protein is present in its normal environment, and therefore (at least at the beginning of the purification procedure) the protein within the source material should exist in its native structure. A second option, used with increasing frequency, is “heterologous expression”, in which the protein of interest is expressed from coding DNA inserted into a host organism. As with natural sources, heterologous expression has advantages and disadvantages. Advantages include the potential for production of the protein in high yield and the possibility of experimentally manipulating the protein sequence prior to synthesis of the protein. Heterologous expression involves a greater investment of time and resources in generating the source in the first place (the host organism must be engineered to produce the protein of interest, and then must be grown in the laboratory). Heterologous expression also involves the possibility that the internal environment of the host organism may lack features required for proper synthesis of the protein. This is especially true for bacterial systems that generally are incapable of incorporating the posttranslational modifications found in many eukaryotic proteins. Two major types of systems are used for heterologous protein expression: microorganisms and multicellular organisms. Bacterial systems are the most commonly used source of heterologous protein production. Bacteria have several advantages, including high levels of expression, rapid, inexpensive growth, and amenability to manipulation. Bacterial systems, however, also have some disadvantages. Bacteria are prokaryotes, and their intracellular environment is somewhat different from that in eukaryotes. Differences in intracellular environment and differences in molecular chaperone content may prevent the heterologous protein from folding. In addition, posttranslational modifications (especially glycosylation, but also proteolytic cleavage and other modifications) will either not be performed or may be performed differently in bacteria than in eukaryotic organisms. Yeast grow faster than other eukaryotes, and perform some types of eukaryotic post-translational modifications. However, yeast are more difficult to grow than bacteria, generally result in lower protein expression levels than bacteria, and are difficult to disrupt when attempting to release the expressed protein. Higher eukaryotic cells (such as insect cells or mammalian cells) in culture can also be used to express proteins. The intracellular environments in these cells are much more similar to those found in the natural source. However, cells of higher eukaryotes grow slowly and are expensive to culture. In addition, eukaryotic cells tend to have very low expression levels. They are therefore used largely when the other systems are not likely to work. Finally, multicellular organisms (both plants and animals) can be engineered to overexpress proteins. Engineering multicellular organisms is an expensive and time-consuming process rarely used except for proteins of considerable commercial value. Protein purification When attempting to understand how a protein works, it is usually necessary to isolate the protein from other proteins that are present in the tissue. This allows the protein to be studied with some assurance that the results reflect the protein of interest and are not due to other molecules that were originally present in the tissue. In addition, some of the techniques used to study proteins will not yield interpretable results unless the protein preparation is homogeneous. Protein purification is therefore a commonly used biochemical technique. Most proteins are fairly large molecules. They are smaller than DNA molecules, but they are tremendously large when compared to the molecules typically used in organic chemistry. The three-dimensional structure of most proteins is a consequence of many relatively weak non-covalent interactions. Disrupting this three-dimensional structure, on which the function of the protein depends, is therefore a relatively easy process. Conversely, preventing the loss of the noncovalent structure (and sometimes the covalent structure) is frequently difficult. Disrupting cellular structure is required to release the proteins from the cell. However, the process has two side effects that may damage proteins: 1) cell disruption typically involves shearing forces and heat, both of which can damage proteins, and 2) cells normally contain proteases (enzymes that hydrolyze other proteins). In most cells, proteases are carefully controlled; however, disruption of the cell usually also releases the proteases from their control systems, and may allow the cleavage of the protein of interest. Purification of proteins involves taking advantage of sometimes-subtle differences between the protein of interest and the remaining proteins present in the mixture. Because proteins are all polymers of the same twenty amino acids, the differences in properties tend to be fairly small. In most cases, current understanding of protein structural and chemical properties is insufficient to allow a purification method to be generated theoretically. The “Art” in the title of this section reflects the fact that development of most protein purification procedures is a matter of trial and error. The table below lists some of the general properties of proteins that can be useful for protein purification, and some of the methods that take advantage of these properties. Each of these general methods will be discussed in some detail below. Note that for any given protein, only some of these methods will be useful, and therefore protein purification schemes vary widely.