title: Beneficial properties of single-domain antibody fragments for application in immunoaffinity purification and immuno-perfusion chromatography.
abstract: We explored the possibility to apply single-domain antibodies from Camelidae for immunoaffinity purification of the ice structuring protein (ISP) from Lolium perenne, which modifies ice crystal growth and therefore has potential application in medicine, biotechnology, agriculture and (frozen) foods. Using phage display together with an appropriate selection method, a group of candidate fragments was isolated from a llama-derived immune library. Affinity chromatography using a purposely selected antibody coupled to a matrix yielded a completely pure and functional ISP. Due to the extreme refolding capabilities and physical stability of single-domain antibodies, the affinity matrix could be regenerated more than 2000 times without loss of capacity, while the fragment's monomeric nature permitted an efficient elution of antigen. The results of this study show that highly pure proteins can be recovered from biological material in a single-step process.
authors.affiliation: Department of Molecular and Cellular Biology, University of Utrecht, Utrecht 3500 TB, The Netherlands. p.verheesen@bio.uu.nl
authors.forename: P
authors.initials: P
authors.lastname: Verheesen
authors.forename: M R
authors.initials: MR
authors.lastname: ten Haaft
authors.forename: N
authors.initials: N
authors.lastname: Lindner
authors.forename: C T
authors.initials: CT
authors.lastname: Verrips
authors.forename: J J W
authors.initials: JJ
authors.lastname: de Haard
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D021301:Antifreeze Proteins / Q000276:immunology / Q000302:isolation & purification*; D002161:Camelids, New World; D002845:Chromatography; D002846:Chromatography, Affinity; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000276:immunology; D008969:Molecular Sequence Data
chemical_list: D021301:Antifreeze Proteins; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region