In the world of biotechnology research, it’s common to use protein engineering techniques when designing or modifying proteins. There are a number of reasons proteins may need to be modified. For example, within the biotech industry, proteins are often used for special applications.
Scientists utilise techniques using high viscosity pumps that allow them to isolate proteins before the purification step begins. This means that it is possible to study substrate specifications and conformations. Beyond that, the way the proteins react to each other is studied. Ligands and activity specific to the enzyme is also evaluated. The term “ligand” is used to describe proteins that are able to attach to receptor proteins.
After the purification process is complete, the level of purity reached will vary based on the needs of the protein that is being worked with. In certain cases, for example, a basic crude extract is more than fine. In other applications, such as the food industry or the pharmaceutical industry, it is necessary to reach a higher level of purification. In these instances, it is usually necessary to undergo multiple purification techniques before the wanted purity level is reached.
Building a Strategy
With each stage of the purification process, the total product content is reduced. Because of this, the best purification plans are the ones that lead to a high purification level with minimal steps. It’s important to keep in mind that the number of steps within a process can vary based on many factors, including the protein size, solubility, and charge. Because of this, the procedures below are better suited for purifying single cytosolic proteins.
For cytosolic protein complexes, it will be necessary to use other techniques.
Readying the Crude Extract
Before intracellular proteins can be purified, the crude extract must be prepared. The term “intracellular proteins” is used to describe proteins that are located within a cell. It’s typically to find a fairly complex protein mixture within crude extracts. These proteins are taken from the cell cytoplasm, as are nutrients, macromolecules, and cofactors.
After the crude extract is fully prepared, it can be utilised for various biotechnology applications. If there is an issue with purity, it will be necessary to go through addition steps. It’s possible to prepare protein extracts by getting rid of cellular debris. This is known as cell lysis and can be done by utilising chemicals, enzymes, sanitation, or a French press.
How Debris Is Removed from Crude Extract
Debris is removed from crude extract via the centrifugation process. Once the process is finished, it’s possible to recover the supernatant. For extracellular preparations, it’s possible to obtain proteins naturally during centrifugation when cells are removed.
Thermostable enzymes are needed for certain biotechnology practices. These are enzymes that are not denatured after being exposed to very high temperatures. With these enzymes, it is possible to maintain higher activity levels.
The Purification Steps for Intermediate Proteins
The majority of modern biotechnical protocols utilise commercially-available kits. In addition to this, it’s standard to use techniques that provide pre-made solutions for basic procedures. Both filters and gel-filtration columns are frequently used during these activities.
Ideally, instructions should be followed with care when working with these kits. When the instructions are followed, you can make sure that you utilise the appropriate amount of solution and follow the correct waiting time. A waiting time is specified to ensure that there is an appropriate concentration of liquid when it is placed in a clean test tube.
Bench-top columns and automated HPLC equipment are used for this technique. In instances where HPLC is used, reverse-phrase, size-exclusion, or ion exchange is utilised. The samples that are obtained can be detected via diode array or laser equipment.