An efficient protein purification system.
Affinity chromatography using affinity tags can be used as a rapid, facile purification separation technique for isolating biologically active compounds, depending upon the affinity properties of the tag rather than those of the target protein. Some tags are relatively small in size (e.g. His-tag), which allows for minimal interaction with the target protein. Nevertheless, these tags fail to provide a highly specific interaction, which leads to low column capacity and sample impurities. Other affinity tags (e.g. maltose binding protein, glutathione-S-transferase) are in some cases relatively large, sometimes even larger than the protein of interest, and thereby might impair protein activity. The cellulosomal dockerin-cohesin interaction may serve as an efficient affinity purification method, however the binding to the immobilized cohesion is only partially reversible upon treatment with EDTA, and only negligible amounts of the target protein are eluted from the affinity column. The present invention allows for an efficient protein purification using a truncated dockerin affinity tag.
Affinity-based protein purification system
Efficient degradation of cellulose by the anaerobic thermophilic bacterium Clostridium thermocellum is carried out by the multi-enzyme cellulosome complex. The enzymes on the complex are attached in a calcium-dependent manner via their dockerin (Doc) module to a cohesin (Coh) module of the cellulosomal scaffoldin subunit. In the present technology, the Coh–Doc interaction was optimized for the purpose of protein affinity purification, by using a shortened Doc of only 48 residues, which is sufficient to function as an effective affinity tag. Thus, Coh–Doc affinity columns provide an efficient and attractive approach for purifying proteins.