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.
Attaching a protein to a bead can be a detailed process that requires forethought and careful planning. Generally, a molecule is attached to a particle through a surface group available on the coating of that particle, for example in the case of streptavidin beads. In cases where the attachment is covalent, it is essential to choose a binding site on the molecule that will allow for proper orientation, maximally presenting the desired site to the sample while still retaining a strong attachment between the molecule and the bead.
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.
Preparing magnetic beads for a particular assay, such as streptavidin beads, requires the beads to be functionalized. The beads need to be attached to the biological material that will serve as a capture molecule in the application. The particular type of attachment by which a molecule is linked to the bead will depend primarily on two things: the molecule being bound and the aim of the process.
Should I use streptavidin beads or gold particles when developing my IVD kits? In order to ensure the success of a protocol, it is essential to have a clear and unbiased knowledge base and a reliable source of reference material. When trying to decide the best platform or application to use for a process, it is critical to ensure that the information on which the decision will be based is generic and factual, and not propagated as promotional data.
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.
The main reason for changing from latex to magnetic latex particles is the need for a change from an homogeneous to an heterogeneous immunoassay. The latter allows washing steps, which potentially help to improve analytical sensitivity and to reduce interference from sample components. The main consideration in shifting from a process that utilizes latex beads to one that uses magnetic latex beads will be the physical separation process itself. Applications that utilize latex beads traditionally make use of a centrifuge or, alternatively, tangential filtration. In contrast, processes that use magnetic latex beads are carried out in a biomagnetic separator. As such, it is necessary to acquire an adequate separator for the process. Ideally, this would be a homogeneous separator.
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.
There are a number of reasons why a lab might want to switch from colloidal gold particles to magnetic beads (for example, streptavidin beads) in an in vitro diagnostic assay, such as swapping from optical to magnetic detection in a lateral flow format or charging the format from lateral flor to a lab automated platform. Making the shift from utilizing one nanoparticle to another, however, is not a straightforward process. There are several areas where problems may arise. There is a considerable difference, for instance, in the way gold particles and magnetic beads bind a molecule. Moreover, the methodology for applications utilizing gold particles is significantly different from that of processes that make use of magnetic beads.
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.
