Beneficial properties of single-domain antibody fragments for application in immunoaffinity purification and immuno-perfusion chromatography P. Verheesen a,b, * , M.R. ten Haaft b , N. Lindner c , C.T. Verrips a,d , J.J.W. de Haard b,1 a Department of Molecular and Cellular Biology, University of Utrecht, Utrecht 3500 TB, The Netherlands b Biotechnology Application Centre BV, Bussum 1400 CA, The Netherlands c Unilever Research and Development, Sharnbrook, Bedford MK44 1LQ, UK d Department of Microbial Biotechnology, Unilever Research, Vlaardingen Laboratory, Olivier van Noortlaan 120, Vlaardingen 3133 AT, The Netherlands Received 31 March 2003; received in revised form 3 September 2003; accepted 5 September 2003 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. D 2003 Elsevier B.V. All rights reserved. Keywords: Single-domain antibody fragment; Phage display; Immunoaffinity chromatography; Ice structuring protein; Protein purification 1. Introduction Affinity chromatography is a powerful technique for the analysis and purification of (bio)molecules. The method relies on the specific high-affinity interaction between a ligand, i.e. the molecule immobilized on a solid support, and a ligate, i.e. the molecule present in a mobile phase. The ligate is captured by the ligand from the solution containing impurities and subsequently the molecule of interest is released from the complex under conditions affecting the interaction. This single-step procedure enables a rapid and efficient purification, whereas traditional methods utilize a series of successive chromatographic separations to achieve the same degree of purity. This study describes the use of immunoaffinity chroma- tography for the purification and quantification of an ice structuring protein (ISP). ISPs, also known as antifreeze proteins [1], exhibit a diversity of functions including depressing freezing temperatures (thermal hysteresis), sup- pressing or modifying ice crystal growth and inhibition of ice recrystallization. This class of proteins naturally occurs in a variety of species including fish, insects and plants [2,3]. The target molecule of this study has been discovered in the over-wintering perennial ryegrass Lolium perenne [4]. Grass ISP has less effect on depressing freezing temper- atures than other known ISPs, but it is a better inhibitor for ice recrystallization [5]. The opportunities for applications of ISPs based on their ability to inhibit ice recrystallization include improvement of storage of medical materials such as blood and organs [6], but many applications could be thought of in the field of the frozen food industry [2,3]. We examined the use of single-domain antibody frag- ments for affinity purification. Animals belonging to the species of Camelidae contain a high fraction of heavy-chain 0304-4165/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2003.09.006 * Corresponding author. Department of Molecular and Cellular Biology, University of Utrecht, Padualaan 8, Utrecht 3584 CH, The Netherlands. Tel.: +31-30-253-5421; fax: +31-30-251-3655. E-mail address: p.verheesen@bio.uu.nl (P. Verheesen). 1 Current address: Ablynx NV, B-9052 Gent, Belgium. www.bba-direct.com Biochimica et Biophysica Acta 1624 (2003) 21 – 28