title: Calcium-sensitive immunoaffinity chromatography: Gentle and highly specific retrieval of a scarce plasma antigen, collectin-LK (CL-LK).
abstract: Immunoaffinity chromatography is a powerful fractionation technique that has become indispensable for protein purification and characterization. However, it is difficult to retrieve bound proteins without using harsh or denaturing elution conditions, and the purification of scarce antigens to homogeneity may be impossible due to contamination with abundant antigens. In this study, we purified the scarce, complement-associated plasma protein complex, collectin LK (CL-LK, complex of collectin liver 1 and kidney 1), by immunoaffinity chromatography using a calcium-sensitive anti-collectin-kidney-1 mAb. This antibody was characterized by binding to CL-LK at hypo- and physiological calcium concentrations and dissociated from CK-LK at hyperphysiological concentrations of calcium. We purified CL-LK from plasma to a purity of 41% and a yield of 38%, resulting in a purification factor of more than 88,000 in a single step. To evaluate the efficiency of this new purification scheme, we purified CL-LK using the same calcium-sensitive mAb in combination with acidic elution buffer and by using calcium-dependent anti-CL-K1 mAbs in combination with EDTA elution buffer. We found that calcium-sensitive immunoaffinity chromatography was superior to the traditional immunoaffinity chromatographies and resulted in a nine-fold improvement of the purification factor. The technique is applicable for the purification of proteins in complex mixtures by single-step fractionation without the denaturation of eluted antigens, and it allows for the purification of scarce proteins that would have otherwise been impossible to purify and, hence, to characterize. This technique may also potentially be applied for the purification of proteins that only interact with calcium ions at hyperphysiological concentrations.
authors: Department of Cancer and Inflammation Research, Institute of Molecular Medicine, University of Southern Denmark, J. B. Winsloews Vej 21.1, DK-5000 Odense, Denmark.
authors: Maiken L
authors: ML
authors: Henriksen
authors: Department of Cancer and Inflammation Research, Institute of Molecular Medicine, University of Southern Denmark, J. B. Winsloews Vej 21.1, DK-5000 Odense, Denmark.
authors: Kirstine L
authors: KL
authors: Madsen
authors: Department of Cancer and Inflammation Research, Institute of Molecular Medicine, University of Southern Denmark, J. B. Winsloews Vej 21.1, DK-5000 Odense, Denmark.
authors: Karsten
authors: K
authors: Skjoedt
authors: Department of Cancer and Inflammation Research, Institute of Molecular Medicine, University of Southern Denmark, J. B. Winsloews Vej 21.1, DK-5000 Odense, Denmark.
authors: Soren
authors: S
authors: Hansen
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry; D002118:Calcium / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods* / Q000592:standards; D037222:Collectins / Q000097:blood* / Q000276:immunology; D006801:Humans; D012680:Sensitivity and Specificity
chemical_list: D000911:Antibodies, Monoclonal; D037222:Collectins; D002118:Calcium
keywords: Collectins; Complement; Immunoaffinity chromatography; Plasma proteins; Purification