Protein engineering methods are largely utilised while performing biotechnology research. This is done while trying to modify or make proteins. There’s a possibility to design and modify proteins if they are required for special uses in the field of biotech.
Scientists used techniques that make sure proteins are isolated before they are actually purified. This is carried out in a drum pump so that the conformations and specifications of the substrate can be studied well and adequately. Besides these, the reactions between the proteins and other ligands are studied together with the enzyme-specific activity. Ligands are defined as proteins that hold themselves onto receptor proteins.
After purification, the degree of purity in the proteins largely depends on the demand of the protein. In some cases, a crude extract is more than enough, while at times this is too little. For other uses like in the food industry and pharmaceuticals, higher purity levels are required. Therefore, various purification activities are required to reach the needed level of purity.
Coming Up With a Strategy
Every step in the process of protein purification reduces the contents of the product. Hence, suitable purification strategies are the ones that lead to the highest levels of purification while involving the fewest steps. But, the steps involved in the process depend on the charge, solubility, size and more properties of the protein used. In such cases, the following steps are best for purifying single cytosolic proteins.
While dealing with cytosolic protein complexes, different techniques have to be used.
Preparing the Crude Extract
The initial important step for purifying intracellular proteins is getting the crude extract ready. Intracellular proteins are those found within the cell. Crude extracts have a complex protein mixture originating from the cell’s cytoplasm plus cofactors, macromolecules and nutrients.
After the crude extract is ready, it’s used in various biotechnology processes. If it’s not pure enough, some steps must be done to rectify this. The protein extracts that are made ready are done by eliminating cellular debris. These usually form as a result of cell lysis; made to happen using chemicals, enzymes, sanitation or French Press.
Eliminating Debris from a Crude Extract
To eliminate debris from the extract, the process of centrifugation needs to be carried out. Once the process is done, the supernatant is recovered successfully. In instances where extracellular preparations are involved, proteins are acquired naturally, during cell removal as centrifugation is being done.
Several biotechnology practices need thermostable enzymes. Thermostable enzymes are those able to stand very high temperatures without getting denatured. These kinds of enzymes also maintain a high activity level.
Intermediate Protein Purification Steps
Up to now, many up-to-date biotechnical protocols have utilised kits that can be found on the commercial level. Still, they’ve also utilised techniques that provide more solutions for a majority of the standard procedures. Gel-filtration columns as well as filters are mostly used while performing protein purification processes.
While working with a dialysis kit, it’s useful to follow the instruction on the kit. The instructions provide a guide to use the right amount of solution plus proper waiting time. The kits have a specific waiting time so that the liquid can be of appropriate concentration when it gets to the clean test tube.
The method is done using either bench-top columns or automated HPLC equipment. When the HPLC is involved, the reverse-phase, ion change or size-exclusion method is used. Diode array or laser technology is used to detect the samples obtained.