Engineering a reversible, high‐affinity system for efficient protein purification based on the cohesin–dockerin interaction

Abstract

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 this study, we have optimized the Coh-Doc interaction for the purpose of protein affinity purification. A C. thermocellum Coh module was thus fused to a carbohydrate-binding module, and the resultant fusion protein was applied directly onto beaded cellulose, thereby serving as a non-covalent "activation" procedure. A complementary Doc module was then fused to a model protein target: xylanase T-6 from Geobacillus stearothermophilus. However, the binding to the immobilized Coh was only partially reversible upon treatment with EDTA, and only negligible amounts of the target protein were eluted from the affinity column. In order to improve protein elution, a series of truncated Docs were designed in which the calcium-coordinating function was impaired without appreciably affecting high-affinity binding to Coh. A shortened Doc of only 48 residues was sufficient to function as an effective affinity tag, and highly purified target protein was achieved directly from crude cell extracts in a single step with near-quantitative recovery of the target protein. Effective EDTA-mediated elution of the sequestered protein from the column was the key step of the procedure. The affinity column was reusable and maintained very high levels of capacity upon repeated rounds of loading and elution. Reusable Coh-Doc affinity columns thus provide an efficient and attractive approach for purifying proteins in high yield by modifying the calcium-binding loop of the Doc module.

Unique Contribution of the Cell Wall-Binding Endoglucanase G to the Cellulolytic Complex in Clostridium cellulovorans

“…Nevertheless, it is possible that the different binding affinities originated from the dockerin sequence of EngG. The sequence duplication of the dockerin module is reflected in its near-perfect 2-fold structural symmetry (8,32). The bidirectional binding of dockerin is termed the "dualbinding mode."…”

“…The recognition between cohesins and dockerins is mediated mainly by hydrophobic interactions. These tight protein-protein interactions allow the integration of the cellulosomal enzymes into the complex (8). In C. cellulovorans, the bacterial surface layer homology (SLH) domains in the scaffolding proteins mediate binding between the cellulosome and the cell surface (4).…”

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

“…All cloning was based on previously described vectors (Karpol et al, 2008(Karpol et al, , 2009. These vectors contained a G. stearothermophilus xylanase T-6 (Xyn) fused to a C. thermocellum Cel48S dockerin in its wild-type (wtDoc) or truncated form (DDoc) (at the C-or N-terminus).…”

“…A schematic representation of the general approach based on the high-affinity cohesin-dockerin interaction can be found in a previous report (see Figure 1, Karpol et al, 2009). CBM-Coh is first bound to the beaded cellulose resin, followed by a sample containing the desired DDoc-bearing target protein in the presence of Ca 2þ .…”

“…These properties suggested that the DDoc would be particularly appropriate for use as an affinity tag in affinity chromatographic applications. Indeed, the utility of this tag was demonstrated in the purification of G. stearothermophilus XynT-6 as a model target protein (Karpol et al, 2009).…”