Purification of Histidine-Tagged Single-Chain Fv-Antibody Fragments by Metal Chelate Affinity Precipitation Using Thermoresponsive Copolymers Ashok Kumar, 1 Per-Olof Wahlund, 1 Cecilia Kepka, 2 Igor Yu Galaev, 1 Bo Mattiasson 1 1 Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-22100, Lund, Sweden; telephone: +46 46 2228264; fax: +46 46 2224713; e-mail: Bo.Mattiasson@ biotek.lu.se 2 Department of Biochemistry, Lund University, Lund, Sweden Received 30 January 2003; accepted 17 July 2003 Published online 11 September 2003 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.10810 Abstract: Metal chelate affinity precipitation (MCAP) has been successfully developed as a simple purification process for proteins that have affinity for metal ions. The present lack of widespread applications for this technique as compared to immobilized metal affinity chromatography (IMAC) may be related to the scarcity of well-characterized metal affinity macroligands (AML) and their applications to the number of different purification systems. In the present work we describe a detailed study of a new purification system using metal-loaded thermoresponsive copolymers as AML. The copolymers of vinylimidazole (VI) with N-isopropylacrylamide (NIPAM) were synthesized by radical polymerization with imidazole contents of 15 and 24 mol%. When loaded with Cu(II) and Ni(II) ions the copolymers selectively precipitated extracellularly ex- pressed histidine-tagged single-chain Fv-antibody frag- ments (His 6 -scFv fragments) from the fermentation broth free from E. coli cells. Precipitation was induced by salt at mild temperatures and the bound antibody fragments were recovered by dissolving the protein – polymer complex in EDTA buffer and subsequent reprecipitation of the poly- mer. His 6 -scFv fragments were purified with yields of 91 and 80% and purification folds of 16 and 21 when Cu(II) and Ni(II) copolymers were used, respectively. The protein precipitation capacity of the Ni(II) copolymer showed a dependence on the VI concentration in the copolymer. The SDS-PAGE pattern showed significant purification of the antibody fragments. B 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 494 – 503, 2003. Keywords: metal chelate affinity precipitation; His 6 -scFv fragments; thermore-sponsive copolymers; affinity macro- ligands; thermoprecipitation; bioseparation INTRODUCTION Genetic engineering has facilitated the large-scale produc- tion of new proteins and peptides (Paul, 1981) and the rapid advancement of such technology has made it possible to obtain practically any desired protein product. The key lies in the recovery of these products with more efficient and economical downstream processes. Thus, the development of efficient and fast purification protocols in bioseparation has always been a challenging task. Affinity techniques for protein purification provide a means of purifying a specific protein from a complex mixture. The concept of using metal chelates in affinity techniques constituted a breakthrough (Porath et al., 1975). Immobi- lized metal affinity (IMA) techniques e.g., IMA-chroma- tography (IMAC), are now being widely applied in protein purification, particularly when dealing with recombinant proteins (see reviews by Chaga, 2001; Gabrec-Porekar and Menart, 2001). At present, it is one of the most popular and successful methods used for the purification of recombinant proteins. The technique offers a number of important advantages over other biospecific affinity tech- niques for protein purification, particularly with respect to ligand stability, binding capacity, protein recovery, and matrix regeneration (Arnold, 1991). The technique is based on the selective interaction between the electron donor groups on the proteins and on the transition-metal ions [Cu(II), Ni(II), Zn(II) or Co(II)] which are loaded on the chelating ligands coupled to a solid support. The amino acids histidine, cysteine, tryptophan, and arginine have strong electron donor groups in their side chains and the presence of such exposed residues is an important factor for IMA-binding properties. However, the actual protein B 2003 Wiley Periodicals, Inc. Correspondence to: Bo Mattiasson Contract grant sponsors: Swedish Competence Center for Bioseparation (CBioSep); INTAS Contract grant numbers: INTAS #0057 & 0673