Characterization of a dockerin-based affinity tag: application for purification of a broad variety of target proteins

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 Ca2+-dependent interaction between the enzyme-borne dockerin and the scaffoldin-borne cohesin modules. In this study, we characterized a new protein affinity tag, Delta Doc, a truncated version (48 residues) of the Clostridium thermocellum CeI48S dockerin. The truncated dockerin tag has a binding affinity (K-A) of 7.7 x 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 beta-glucosidase, Escherichia toll 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 beta-glucosidase, purified using this approach, was tested and found to be similar to that of a beta-glucosidase preparation (without the Delta Doc 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 Ca2+-binding loop of the dockerin module results in an efficient and robust affinity tag that can be generally applied for protein purification. Copyright (C) 2010 John Wiley & Sons, Ltd.