Protein engineering techniques are widely used in biotechnology research. This is done in an effort to modify or design proteins. Proteins can be designed and modified if they are needed for special applications relating to the biotech industry.
The techniques used by scientists ensure that proteins are isolated before they undergo the purification process. This is done so that proper studies can be carried out on the conformations and the specifications of the substrate. In addition, the reactions between proteins and other ligands are studied along with the enzyme-specific activity. Ligands are referred to as proteins that attach to special receptor proteins.
When proteins are purified, the degree of purity mainly depends on the demand of the protein in question. In some instances, a simple crude extract is sufficient. For other uses such as pharmaceuticals and in the food industry, a higher level of purification is needed. Hence, several purification techniques are needed to attain the desired purity level.
Developing a Strategy
Each step in the protein purification process reduces product content. Hence, ideal purification strategies are those which result in the highest purification level with the least number of steps. However, the steps used in the process are dependent on the solubility, charge, size, and other properties of the protein used. As such, the following procedures are more suited for purifying proteins that are single cytosolic.
In the case of cytosolic protein complexes, these require different techniques.
Preparing the Crude Extract
The first major step for purifying intracellular proteins is preparing the crude extract. Intracellular proteins are those that exist inside of the cell. Crude extracts usually contain a very complex mixture of proteins that come from the cytoplasm of the cell along with cofactors, nutrients, and macromolecules.
When the crude extract has been prepared, it is used for several biotechnology applications. If purity becomes an issue, a set of subsequent steps must be undertaken. The protein extracts which are prepared are done by removing cellular debris. These are created by cell lysis; this is accomplished with the use of enzymes, chemicals, a French Press, or sonication.
Removing Debris from a Crude Extract
In order to remove the debris from the extract, it must undergo the process of centrifugation. After the process is completed, the supernatant is successfully recovered. In the case of extracellular preparations, proteins are naturally obtained when the cells are removed during the centrifugation process.
Some biotechnology practices require thermostable enzymes. Thermostable enzymes are those which can withstand the highest temperatures without being denatured. Thermostable enzymes also maintain a high level of activity.
Intermediate Protein Purification Steps
Thus far, most modern biotechnical protocols have made use of kits that are available on a commercial level. Additionally, they have also used techniques that offer a more ready-made solution for most standard procedures. Gel-filtration columns and filters are often used while conducting protein purification activities.
Dialysis Kit
When using a dialysis kit, it’s best to follow the direct instructions provided by the kit. The instructions ensure that the correct amount of solution is used followed by the appropriate waiting time. The kits specify a waiting time so that the eluant is of the right concentration when it enters the clean test tube.
Chromatographic Methods
This method is carried out with the use of either automated HPLC equipment or bench-top columns. When using the HPLC, ion exchange, reverse-phase or the size-exclusion method is used. Samples obtained are detected with laser technology or diode array.
Immunoblotting
This is the process that applies affinity chromatography along with a special protein visualization technique. Ligands are defined by the antibodies of specific proteins on the chromatography column. When the target protein sticks to the column, it can be removed by rinsing the column with a normal salt solution. After the target protein has been separated from the mixture, dye labels or radioactive antibodies aid in their detection.
Learn more about antibody-enzyme conjugates.
