Proteins are of interest for their nutritious functions and health benefits to human. Protein separation/purification is highly demanded in biomedicine, bioscience and other fields. Separation based on magnetic beads (MBs) is an old but also an emerging technique that possesses several advantages compared with traditional techniques like chromatography, precipitation, ultrafiltration, centrifugation, and dialysis. Generally, MBs for protein purification are often immobilized with ligands (e.g. affinity ligands, pseudo affinity ligand, or ion-exchange groups), which have been proven to have affinity to target protein.
Principles of Magnetic Beads (MBs) Protein Purification
In general, MBs based protein purification mainly depends on the principle of magnetic affinity separation, including direct and indirect methods.
Direct Method
Through physical adsorption and covalent bonding, the specific proteins/antibodies are directly coupled to the magnetic beads, and then combined with the corresponding antigens to form an “antigen-antibody-MBs” complex. This complex can not only directly target to the specific substance, but also shows high magnetic responsiveness. Under the action of magnetic force, the compound can move directionally, thereby achieving the purpose of separation, concentration and purification. The direct method has advantages of rapidness, simplicity, strong specificity and high yield, but its sensitivity is low, and it is necessary to prepare corresponding antibody-conjugated MBs.
Fig. 1 Schematic illustration of direct magnetic affinity purification. (Trends in Food Science & Technology 2012, 27 (1), 47-56)
- Indirect Method
The indirect method is to add the free affinity ligand (in most cases an appropriate antibody) to a solution or suspension to interact with the target compound, and then capture the resulting complex by appropriate MBs. In the case of using antibodies as free affinity ligands, MBs with immobilized secondary antibodies such as protein A or protein G are used to capture the complex. Alternatively, the free affinity ligands can be biotinylated and thus MBs with immobilized streptavidin or avidin are used to capture the complex formed.
The performance of these two methods are equally good, but, in fact, the direct method is easier to control. The indirect method is generally used in the following situations: (i) there are no direct-labeled MBs antibodies; (ii) several antibodies are needed to remove a variety of cells; (iii) the expression level of specific antigen molecules on the target cell is low.
Advantages
- This purification process is simple and only requires a few handling steps. All steps can be performed in one single test tube or another vessel. This technology does not require complicated chromatography systems, centrifuges, filters or other equipment.
- There is no limitation on the clarity of samples. The separation process can be performed directly in crude samples. It only requires a simple magnetic adsorption step to easily separate the protein/antibody from the expression product, effectively avoiding the shortcomings of traditional chromatography technology for protein/antibody purification.
- Due to the magnetic properties of magnetic adsorbents, they can be relatively easily and selectively removed from the sample. In fact, magnetic separation is the only feasible method for recovery of small magnetic particles (diameter ca 0.1 ~ 1 μm) in the presence of biological debris and other fouling material of similar size.
- The magnetic separation techniques are also the basis of various automated procedures, especially magnetic particle-based immunoassay systems for the determination of a variety of analytes. Several automated systems for the separation of proteins or nucleic acids have become available recently.
- Furthermore, the MBs purification technology also has the advantages of good cost-effectiveness, high sensitivity, high specificity, fast detection speed and good repeatability.
Available MBs Products for Protein Purification
1. Protein A-MBs
Recombinant protein A covalently attached to the surface of the blocked MBs can bind antibodies from many different species, enabling the purification of antibodies from crude extracts. The immunoprecipitation assay with protein A-coated MBs has low background and high yield of target antigen. Protein A-MBs are usually used to separate antibodies from serum, cell culture supernatant or ascites fluid, and used for immunoprecipitation and co-immunoprecipitation of antigens in cell or tissue extracts.
2. Protein A/G-MBs
The recombinant protein A/G immobilized on MBs is a fusion of the IgG binding domain of protein A and protein G. Therefore, protein A/G-MBs is not only a mixture of protein A and protein G alone, but also a mixture of protein A-MBs and protein G-MBs. This makes it possible to capture a wider range of species and isotypes of antibodies, rather than capturing any protein alone.
3. Protein G-MBs
Protein G-MBs contains recombinant protein G, and each protein has two Fc binding domains. These domains can bind antibodies from many different species, including mouse, human, rabbit, cow, goat, and sheep.
4. Protein L-MBs
Protein L-MBs contains recombinant protein L, and each protein has 4 immunoglobulin binding domains. These domains can bind to a wider range of Ig classes through the kappa light chain and protein A or protein G (including IgG, IgM, IgA, IgE and IgD). Protein L also binds to single-chain variable fragments (scFv) and Fab fragments containing kappa light chains. When using complex biological samples, the proprietary blocking agent on the magnetic beads can minimize or eliminate non-specific binding on the surface of the beads.
5. His-tag protein purification MBs
The agarose microspheres of His-tag protein purification MBs are filled with magnetic cores, and metal ions Ni2+ or Co2+ are fixed on the surface. Ni2+ or Co2+ can form a coordination bond with histidine in the His tag of the protein, bind the target protein to the magnetic beads, and finally separate it by a magnetic separator. High-purity target protein is purified in one step, which significantly simplifies the purification process and reduces equipment requirements. It is suitable for the rapid and convenient purification of histidine-tagged protein in scientific research and industrial fields.
6. GST fusion protein purification MBs
It is a new type of functional material designed for efficient and rapid purification of glutathione S-transferase (GST) fusion protein. It can directly purify high-purity target protein from biological samples through magnetic separation.
7. Strep-tag II protein purification MBs
It is a new type of functional material designed for efficient and rapid purification of Strep-tag II protein. Strep-tag II only consists of 8 short peptides with chemical balance (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys), so its influence on the activity of the recombinant protein is negligible. During the purification process, there is no need to cut Strep-tag II in the target protein. At the same time, the target protein can be purified under physiological conditions. Compared with other tags, these mild purification parameters can preserve the biological activity of the protein and obtain the target protein with high purity.
References
1. Cao, M.; Li, Z.; et al. Food related applications of magnetic iron oxide nanoparticles: Enzyme immobilization, protein purification, and food analysis. Trends in Food Science & Technology 2012, 27 (1), 47-56.
2. Safarik, I.; Safarikova, M., Magnetic techniques for the isolation and purification of proteins and peptides. BioMagnetic Research and Technology 2004, 2 (1), 7.
3. Franzreb, M.; Siemann-Herzberg, M.; et al. Protein purification using magnetic adsorbent particles. Applied microbiology and biotechnology 2006, 70 (5), 505-516.