As you will find in our protein purification handbook, choosing the right matrix for protein enrichment and purification processes, such as immunoprecipitation or pull-down assays, is an important step in optimizing the efficiency of a protocol. The decision will be based on a number of variables and ultimately depends largely on the nature of the target biomolecule. The goal is to choose a matrix that will not only maximize the final yield, but will also be practical and accessible.
It is well known that most purification processes of recombinant proteins are conducted through chromatographies of different types, mainly in column chromatography. Whether it’s by affinity chromatography, gel filtration or ion exchange chromatography, these already established methods are not exempt from limitations. Research in recent years has allowed us to develop a series of alternatives to chromatography that allow us to avoid many of these limitations.
In purification of recombinant proteins, highly pure samples are rarely obtained with the initial stages of the process. Whether we perform a highly specific affinity chromatography (with histidine tags, for example) or purification with several stages of capture and intermediate purification, there are always contaminants in the final sample. As you will find in our protein purification handbook, these contaminants are molecules that are closely related to the protein to be purified since, if a high-resolution technique is not applied, they can be hardly differentiated from the protein to be purified.
Affinity chromatography allows us to obtain good results and a high level of purity with a single purification step, since a structure that is exclusively found in the recombinant protein is used as a tag. However, this is not possible in all cases. There are proteins that don’t accept changes in their sequence, even if the changes are so minimal as the incorporation of a tag, since the proteins quickly lose their biological activity with any modifications in their structure.
The purification of recombinant proteins is a long and complex process, as our protein purification handbook proves, and is influenced by a multitude of variables. As has already been mentioned, the nature of the protein itself, its properties and the characteristics of the host producer will greatly influence the design and development of this procedure. There are also very few available techniques, although it is very well known that the preferred one is affinity chromatography.
During the sample preparation for the purification of recombinant proteins, there are several considerations that will influence its development. All preparation steps, which are thoroughly explained in our protein purification handbook, are important and we have to pay attention to them.
Chromatography is one of the most common methods for the purification of recombinant proteins, and more specifically affinity chromatography is the one that is mostly used due to its high specificity, which allows us to obtain great purity in one single step. When using this technique, a tag is added to the protein of interest, a small structure that is not included in the original protein and that allows us to easily capture it. As we explain in our protein purification handbook, tags are generally short sequences of 3-4 amino acids (up to a maximum of 15) and are intended to minimize as much as possible the properties of the protein.
Recombinant proteins are produced in the host cells along with a great variety of molecules that it contains naturally. However, for most of their applications (such as for example in therapeutics), a recombinant protein should be purified and isolated from the rest of cell molecules. As we explain in our protein purification handbook, this is obtained by the recombinant protein purification processes.
The host in which a recombinant protein is produced doesn’t naturally include the gene of this protein in its genome. Therefore, this gene needs to be introduced in a process called molecular cloning. Successful cloning of a gene requires several elements, which are discussed in our protein purification handbook.
As you can learn in our protein purification handbook, production techniques of recombinant proteins offer multiple options when it comes to available hosts; in other words, the organisms that incorporate the gene of the protein of interest and express it correctly.