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.
Karpol
Alon
A
Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100 Israel.
Kantorovich
Lia
L
Demishtein
Alik
A
Barak
Yoav
Y
Morag
Ely
E
Lamed
Raphael
R
Bayer
Edward A
EA
D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism; D002846:Chromatography, Affinity; D002868:Chromosomal Proteins, Non-Histone / Q000302:isolation & purification* / Q000378:metabolism*; D048013:Acetivibrio thermocellus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D005818:Genetic Engineering; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014995:Xylosidases / Q000378:metabolism; D000097722:Cohesins
D002118:Calcium; D018797:Cell Cycle Proteins; D002482:Cellulose; D002868:Chromosomal Proteins, Non-Histone; D004492:Edetic Acid; D011994:Recombinant Proteins; D014995:Xylosidases; D000097722:Cohesins