Purification Systems Based on Bacterial Surface Proteins
نویسندگان
چکیده
Affinity purification is based on the selective and reversible interaction between two binding partners, of which one is bound to a chromatography matrix and the other may be either a native target protein or a recombinant protein fused with an affinity tag (Cuatrecasas et al. 1968). Recombinant DNA-technology allows straightforward construction of gene fusions to provide fusion proteins with two or more functions. The main intention is to facilitate downstream purification; however gene fusions may also improve solubility and proteolytic stability and assist in refolding (Waugh 2005). There are many fusion partners for which commercially available purification systems exist, ranging in size from a few amino acids to whole proteins (Flaschel & Friehs 1993; Terpe 2003). A commonly used purification handle is the poly-histidine (His) tag, enabling purification of the recombinant protein on a column with immobilized metal ions (Hochuli et al. 1988). Other commonly used tags include the FLAG peptide (binding to anti-FLAG monoclonal antibodies), the strep-tag (binding to streptavidin), glutathione S-transferase (binding to glutathione) and maltose binding protein (binding to amylose) (Terpe 2003). Many affinity chromatography strategies also exist for the purification of native proteins, however these are slightly less specific and generally purify classes of proteins, as individual proteins each need a specific ligand. Today, many different ligands are available that can separate specific groups of proteins, for example phosphorylated, glycosylated or ubiquitinylated proteins (Azarkan et al. 2007).
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