Abstract
Cellulose, a major component of plant matter, is degraded by a cell surface multiprotein complex called the cellulosome produced by several anaerobic bacteria. This complex coordinates the assembly of different glycoside hydrolases, via a high‐affinity Ca^2+^‐dependent interaction between the enzyme‐borne dockerin and the scaffoldin‐borne cohesin modules. In this study, we characterized a new protein affinity tag, ΔDoc, a truncated version (48 residues) of the Clostridium thermocellum Cel48S dockerin. The truncated dockerin tag has a binding affinity (K~A~) of 7.7 × 10^8^ M^−1^, calculated by a competitive enzyme‐linked assay system. In order to examine whether the tag can be used for general application in affinity chromatography, it was fused to a range of target proteins, including Aequorea victoria green fluorescent protein (GFP), C. thermocellum β‐glucosidase, Escherichia coli thioesterase/protease I (TEP1), and the antibody‐binding ZZ‐domain from Staphylococcus aureus protein A. The results of this study significantly extend initial studies performed using the Geobacillus stearothermophilus xylanase T‐6 as a model system. In addition, the enzymatic activity of a C. thermocellum β‐glucosidase, purified using this approach, was tested and found to be similar to that of a β‐glucosidase preparation (without the ΔDoc tag) purified using the standard His‐tag. The truncated dockerin derivative functioned as an effective affinity tag through specific interaction with a cognate cohesin, and highly purified target proteins were obtained in a single step directly from crude cell extracts. The relatively inexpensive beaded cellulose‐based affinity column was reusable and maintained high capacity after each cycle. This study demonstrates that deletion into the first Ca^2+^‐binding loop of the dockerin module results in an efficient and robust affinity tag that can be generally applied for protein purification. Copyright © 2010 John Wiley & Sons, Ltd.