Immunoprecipitation And Related Techniques

These methods are used to physically separate a particular protein from a cell extract. They rely on a high-affinity, sequence-specific interaction between the protein and another molecule capable of specifically binding to that protein, such as an antibody. To achieve physical separation, the molecule providing the recognition specificity is bound to an inert substrate that can be separated physically from the binding reaction mix. Once purified by one of these methods, the protein can be characterized further or its level of expression determined, often by Western analysis. Moreover, these methods can be scaled up for large samples and can thus be used for protein purification.

IMMUNOPRECIPITATION

Immunoprecipitation uses protein- or epitope-specific antibodies for protein recognition. The complex is then removed from the mixture because it is bound to small beads, usually composed of Sepharose (a form of starch), that are themselves bound to protein A, protein G, or a mixture of both. Protein A is a product of the bacterium Staphylococcus aureus and binds to immunoglobulin constant region with very high affinity. Protein G is also a product from a Streptococcus bacterial species and also binds to IgG constant region. Proteins A and G differ slightly in their affinity for the IgG constant region of the different species commonly used to produce antibodies, like horse, mouse, or goat. Thus, researchers often use Sepha-rose beads bound with a mixture of proteins A and G in order to achieve high-affinity binding to a broad range of antibodies that they might be using in different experiments.

An antibody specific to the protein of interest is added to the cell extract (prepared under the appropriate conditions) and allowed to bind. Protein A/G-bound Sepharose beads are added and incubated to allow binding of the antibody-protein complex to the beads. Centrifugation is used to pellet the beads with the bound protein. The sample is then analyzed by Western analysis. If a protein-specific antibody is not easily available but the gene encoding the protein is, then researchers often choose to epitope-tag the protein of interest using the methods described above.

Immunoprecipitation is most often used to identify other proteins that may be found complexed with the protein of interest. For this, the cell extract must be prepared under nondenaturing conditions. Any proteins that interact with the protein of interest should coimmunoprecipitate (so-called co-IP). For example, if one's genetic analysis indicates that proteins X and Y form a complex, then protein Y should co-IP with protein X when anti-protein X antibody is used. Western analysis of the pelleted sample should detect protein X and, if the two proteins co-IP, should also be able to detect protein Y when anti-protein Y antibody is used as the probe.

METAL CHELATE AFFINITY PURIFICATION

Many natural proteins have metal binding sites for ions such as Ni2+ and Zn2+. Metal chelate affinity purification makes use of this to purify proteins. If the protein of interest does not contain a metal binding site, then one can be added by epitope-tagging. By far the most common metal chelate tag is a series of six histidine residues that specifically binds to Ni2+ ions and is referred to as a His-tag. The His-tag is short enough to be inserted by PCR-based methods, but a variety of vectors are available commercially for the construction of His-tagged alleles of the protein of interest.

Metal chelate affinity purification uses resin beads to which a metal chelating group has been bound. Ni2+ ions are then bound to this chelating group in such a way as to allow the Ni2+ ion also to be available for binding by the metal binding group of the protein, the His-tag of the protein of interest. The Ni2+-bound resin beads can be used either as a slurry or packed in an affinity column. First, the His-tagged protein is allowed to bind to the Ni2+-bound resin by incubating the resin with cell extract. After washing off all the unbound protein, the His-tagged protein is released from the beads using an excess of imidazole (an analogue of histidine).

Proteins purified in this manner from cell extracts are frequently contaminated with other cellular proteins that normally contain Ni2+ binding sites. Snfl kinase of Saccharomyces is an example. Therefore, to obtain a strictly pure product one will have to carry out a second purification step. This may not be necessary for many of the characterization methods to be undertaken in follow-up studies, particularly

Western analysis that can detect the protein of interest alone by the use of specific antibodies.

GST-TAGGED AND MalB-TAGGED PROTEINS

Other affinity purification methods are available and can also be used like immuno-precipitation for studies of protein complexes or for protein purification. Two of the most common affinity purification methods use GST and MalB protein fusions. GST is glutatione S-transferase, is a product of Schistosoma japonicum and binds glutathione with high affinity. GST fusions to the protein of interest can be made by standard epitope-tagging methods and many fusion vectors are commercially available to simplify these constructions. Cell extract containing the GST-tagged fusion protein is passed over a resin-bound glutathione affinity column and immobilized. The GST portion is cleaved from the protein of interest with enterokinase or thrombin both of which act on the sequence at the fusion junction site. This releases purified protein from the resin along with any associated proteins.

MalB protein is an E. coli product involved in maltose transport. It is localized to the periplasmic space and is used as a maltose carrier. Its high-affinity binding to maltose and amylose is the basis of this purification method. Using methods such as those described above, one constructs an in-frame fusion of the protein of interest to MalB. Vectors for these constructions are commercially available. The MalB portion will provide specific binding to a column-bound amylose and the MalB fusion protein will be retained by the column. The fusion protein is released from the amylose column by excess maltose. The purified fusion protein is then cleaved at the junction site with a site-specific protease and the protein of interest further purified by passage over an amylose column to remove the free MalB protein and any uncleaved residue.

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