One of the main protein engineering techniques within the biotechnology research world comes from the modification and design of proteins. However, the proteins must firstly be purified to optimise their properties for special applications within the industry. Scientists are generally required to isolate and purify them. Assay development services can be used for specific research.
This is done to ensure that the proteins substrate and conformations specs can be thoroughly studied. In addition, studies are also conducted to determine their reactions with other ligands and protein-specific enzyme activities. Ligands are those proteins that attach to receptor proteins.
To purify a protein, the intended use must first be determined. In some instances, crude extracts are more than sufficient to get the job done. However, in the case of pharmaceuticals and foods, higher levels of purification are needed. Protein purity levels are achieved by using special protein purification techniques.
Developing a Strategy
During the protein purification process, some of the original product is lost. Hence, the most ideal purification strategy ensures that the process is carried out successfully in fewer steps. Selecting the steps to be used is dependent on the charge, the size, the solubility and many other properties of the protein in question. In the case of single cytosolic protein, the following are used:
(It should be noted that the purification of cytosolic protein is far more complex and requires a different method of application.)
Preparing a Crude Extract
When purifying intracellular or proteins that exist inside of the cell, the crude extract must first be prepared. The extract usually contains a specially formulated mixture that comes from within the cytoplasm. The mixture also contains cofactors, nutrients and additional macromolecules that are all essential during the process.
While crude extracts can be used in some biotechnological applications, purification generally becomes an issue and a set of specific steps must be completed. Extracts are usually prepared by removing any debris that was formed by cell lysis. This is achieved with the use of enzymes, chemicals, a French Press or sonication.
Removing Debris From within the Extract
Extract debris is removed via centrifugation. The liquid generated or supernatant on top of the solid residue is then recovered. In the case of extracellular prep, proteins are sourced during centrifugation when the cells are removed. Certain industry-specific applications require enzymes that can handle high temperatures known as thermostable enzymes.
These enzymes maintain their high activity while resisting the denaturing effect by high temperatures.
Intermediate Protein Purification Steps
Advanced biotechnological protocols often benefit from commercially prepared kits and methods that offer premade solutions. Hence purification is conducted with the use of filters and gel filtered columns.
The instructions on the dialysis kit should be followed properly, to ensure that the correct amount of solution and volume are used. It’s also important to wait for the allotted time to collect the eluant in a clean test tube.
These methods can be applied with either the automated HPLC equipment or bench-top columns. Separation via the HPLC is done using ion-exchange, reverse-phase, or the size exclusion method. Samples obtained are then detected using laser tech or diode array.
Immunoblotting is combined with chromatography and protein-specific antibodies are used as the source of the ligand in the chromatography column. The protein usually stays on the column which is then rinsed with salt solutions and other agents. Antibodies that are linked to dye labels or radioactivity helps to determine the target protein after it has been separated from the mixture.