Alon Karpol

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, ΔDoc, a truncated version (48 residues) of the Clostridium thermocellum Cel48S dockerin. The truncated dockerin tag has a binding affinity (KA) of 7.7 × 108 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 Ca2+‐binding loop of the dockerin module results in an efficient and robust affinity tag that can be generally applied for protein purification. Copyright

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

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. Copyright

Intramolecular clasp of the cellulosomal Ruminococcus flavefaciens ScaA dockerin module confers structural stability

The cellulosome is a large extracellular multi‐enzyme complex that facilitates the efficient hydrolysis and degradation of crystalline cellulosic substrates. During the course of our studies on the cellulosome of the rumen bacterium Ruminococcus flavefaciens, we focused on the critical ScaA dockerin (ScaADoc), the unique dockerin that incorporates the primary enzyme‐integrating ScaA scaffoldin into the cohesin‐bearing ScaB adaptor scaffoldin. In the absence of a high‐resolution structure of the ScaADoc module, we generated a computational model, and, upon its analysis, we were surprised to discover a putative stacking interaction between an N‐terminal Trp and a C‐terminal Pro, which we termed intramolecular clasp. In order to verify the existence of such an interaction, these residues were mutated to alanine. Circular dichroism spectroscopy, intrinsic tryptophan and ANS fluorescence, and NMR spectroscopy indicated that mutation of these residues has a destabilizing effect on the functional integrity of the Ca2+‐bound form of ScaADoc. Analysis of recently determined dockerin structures from other species revealed the presence of other well‐defined intramolecular clasps, which consist of different types of interactions between selected residues at the dockerin termini. We propose that this thematic interaction may represent a major distinctive structural feature of the dockerin module.

Structural and functional characterization of a novel type‐III dockerin from Ruminococcus flavefaciens

ScaB cohesin binds to ScaADoc by enzyme linked immunosorbent assay (View interaction)

Insights into a type III cohesin–dockerin recognition interface from the cellulose‐degrading bacterium Ruminococcus flavefaciens

Cellulosomes are large multicomponent cellulose‐degrading assemblies found on the surfaces of cellulolytic microorganisms. Often containing hundreds of components, the self‐assembly of cellulosomes is mediated by the ultra‐high‐affinity cohesin–dockerin interaction, which allows them to adopt the complex architectures necessary for degrading recalcitrant cellulose. Better understanding of how the cellulosome assembles and functions and what kinds of structures it adopts will further effort to develop industrial applications of cellulosome components, including their use in bioenergy production. Ruminococcus flavefaciens is a well‐studied anaerobic cellulolytic bacteria found in the intestinal tracts of ruminants and other herbivores. Key to cellulosomal self‐assembly in this bacterium is the dockerin ScaADoc, found on the non‐catalytic structural subunit scaffoldin ScaA, which is responsible for assembling arrays of cellulose‐degrading enzymes. This work expands on previous efforts by conducting a series of binding studies on ScaADoc constructs that contain mutations in their cohesin recognition interface, in order to identify which residues play important roles in binding. Molecular dynamics simulations were employed to gain insight into the structural basis for our findings. A specific residue pair in the first helix of ScaADoc, as well as a glutamate near the C‐terminus, was identified to be essential for cohesin binding. By advancing our understanding of the cohesin binding of ScaADoc, this study serves as a foundation for future work to more fully understand the structural basis of cellulosome assembly in R. flavefaciens. Copyright