Protein Purification Strategies and Preparation of Cell-Free Extracts
An important part of biotechnology research is to use protein engineering techniques to design or modify proteins with optimized properties for specific industrial applications. In order to do this, scientists must be able to isolate and purify proteins of interest so their conformations, substrate specificities, reactions with other ligands, and specific activities can be studied.
The degree of protein purity required depends on the intended end use of the protein. For some applications, a crude extract is sufficient. However, for other uses, such as in foods and pharmaceuticals, a high level of purity is required. In order to achieve this, several protein purification methods are typically used, in a series of purification steps.
Each protein purification step usually results in some degree of product loss. Therefore, an ideal protein purification strategy is one in which the highest level of purification is reached in the fewest steps. The selection of which steps to use is dependent on the size, charge, solubility and other properties of the target protein. The following techniques are most appropriate for purifying a single cytosolic protein. Purification of cytosolic protein complexes is more complicated and usually requires that different methods be applied.
- The first step in purifying intracellular (inside the cell) proteins is preparation of a crude extract. The extract will contain a complex mixture of all the proteins from the cell cytoplasm, and some additional macromolecules, cofactors and nutrients. Crude extract may be used for some applications in biotechnology, however, if purity is an issue, subsequent purification steps must be followed. Crude protein extracts are prepared by removal of cellular debris generated by cell lysis, which is achieved using chemicals and enzymes, sonication or a French Press. The debris are removed by centrifugation and the supernatant is recovered. Crude preparations of extracellular proteins may be obtained by simply removing the cells by centrifugation.
- For certain biotechnology applications, there is a demand for thermostable enzymes: Enzymes that can tolerate high temperatures without denaturing, and while maintaining a high specific activity. Organisms that produce them are sometimes called extremophiles. An easy approach to purifying a heat-resistant protein is to denature the other proteins in the mixture by heating, then cooling the solution (thus allowing the thermostable enzyme to reform or redissolve, if necessary. The denatured proteins can then be removed by centrifugation.
Intermediate Purification Steps
- In the past, a common second step to purifying a protein from a crude extract was by precipitation in a solution with high osmotic strength (i.e. salt solutions). Nucleic acids in the crude extract can be removed by precipitating aggrigates formed with streptomycin sulfate or protamine sulfate. Protein precipitation is usually done using ammonium sulfate as the salt.
Different proteins will precipitate in different concentrations of ammonium sulfate. In general, proteins of higher molecular weight precipitate in lower concentrations of ammonium sulfate. Salt precipitation does not usually lead to a highly purified protein, but can assist in eliminating some unwanted proteins in a mixture and concentrating the sample. Salts in the solution are then removed by dialysis through porous cellulose tubing, filtration, or gel exclusion chromatography.
- Modern biotech protocols often take advantage of the many commercially-available kits that provide ready-made solutions for standard proceedures. Protein purification is often performed using filters and prepared gel filtration columns. All you have to do is follow the instructions and add the right volume of the right solution and wait the specified length of time while collecting the eluant (what comes out the other end of the column) in a fresh test tube.