Biotechnology research makes extensive use of protein engineering techniques such as immunoassay development. This is done, when there is a need to design or modify proteins. The need to modify or design proteins arises from needs in the biotech industry for special applications.
Before proteins are purified, scientists need to use techniques that first isolate the proteins. This allows proper studies to be conducted on the specifications and conformations of the substrate. At the same time, a study is also made of reaction between the proteins and other ligands, as well as any enzyme-specific activity. Ligands are those proteins that attach themselves to other special receptor proteins.
When proteins undergo the purification process, the degree of purity is dependent on the on the protein that is being purified. At times, a simple crude extract will suffice. For use in the food and pharmaceutical industry, however, it is needed to purify the protein to a higher level. To attain this desired level of purity, use may have to be made of several purification techniques.
Product content gets reduced at every step of the process of protein purification. The best purification strategies are those that lead to the highest purification with the minimum number of steps. Properties of the protein being purified, like size, solubility, charge and others will have a bearing on the number of steps required for purification. The procedures that follow here, will be more suitable for the purification of single systolic proteins.
Preparation of the Crude Extract
The preparation of the crude extract is the first step for the purification of intracellular proteins. These are proteins that have their existence inside a cell. You will find a very complex mixture of proteins in the crude extracts that come from a cell’s cytoplasm, and this will also have macromolecules, nutrients, and cofactors.
The preparation of the crude extract allows it to be used for many biotechnology applications. You need to take a a set of steps, subsequently, if purity becomes an issue. Removal of cellular debris is necessary for preparation of protein extracts. Cell lysis helps to create this, and this is done through the use of chemicals, enzymes, sanitation, or a French press.
Crude Extract Debris Removal
For removing debris from extract it has to undergo the centrifugation process. Once this process of centrifugation is completed, recovery of the supernatant is considered successful. Proteins are obtained naturally when the centrifugation process removes cells ,while making extracellular preparations.
In some practices in biotechnology it may be required to use thermostable enzymes. These are enzymes that are able to withstand very high temperatures without leading to their being denatured. These enzymes also allow for maintenance of a high level of activity.
Steps for Intermediate Protein Purification
Till the present, use has been made of commercially available kits for most modern biotechnical protocols. In addition use has also been made of techniques that can offer ready made solutions for standard procedures. For conducting the activities involved in protein purification use is made of filters and gel-filtration.
It is best if, when use is being made of a dialysis kit, that all direct instructions given in the kit are followed. These instructions will ensure that use is made of he correct amount of the solution along with the waiting time that is appropriate. Waiting times are specified in these kits, so that the liquid has the right concentration when it enters a clean test tube.
For carrying out this method, use is made of bench-top columns or of automated HPLC equipment. When HPLC is used, size-exclusion, ion exchange, or reverse phase is the method employed. Detection of samples is done through a diode array or through laser technology.