Bioseparation 7: 185–194, 1999. © 1999 Kluwer Academic Publishers. Printed in the Netherlands. 185 Metal chelate affinity precipitation: a new approach to protein purification A. Kumar, I.Yu. Galaev & B. Mattiasson ∗ Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, Lund S-221 00, Sweden ( ∗ author for correspondence) Received 14 August 1997; accepted 14 May 1998 Key words: metal chelate, affinity precipitation, poly(N-isopropylacrylamide-co-vinylimidazole), thermoprecipita- tion, cloud-point, protein purification, protein inhibitors Abstract Metal chelate affinity precipitation of proteins, a method combining metal–protein interaction and affinity pre- cipitation is being discussed as a selective separation process for proteins. The technique utilizes a flexible soluble–insoluble thermo-responsive polymer with a covalently linked ligand loaded with metal ions. The affinity binding of the target protein varies with different metal ions. Copolymers of N-isopropylacrylamide with 1- vinylimidazole loaded with Cu(II) ions are designed as a potential carriers for affinity purification and proved to be successful for purification of protein inhibitors from a variety of cereals. Introduction Development of efficient and fast purification proto- cols in bioseparation has always been a challenging task. With the rapid advancement of gene technology, it has been possible to get any desired protein product, but the recovery of such products still poses a major problem. Affinity techniques for protein purification provide means to purify a specific protein from a com- plex mixture. The concept of using metal chelates in affinity techniques was a breakthrough introduction (Porath et al., 1975). As such, immobilized metal affinity (IMA) techniques, e.g., IMA-chromatography, are now gaining wide application in protein purifi- cation particularly when dealing with recombinant proteins (Arnold, 1991; Sulkowski, 1985). This offers a number of important advantages over other ’biospe- cific’ affinity techniques for protein purification partic- ularly with respect to ligand stability, protein loading and recovery (Arnold, 1991). The technique is gener- ally based on the selective interaction between metal ions (Cu(II), Zn(II), Ni(II) or Co(II)) which are fixed on the solid support and electron donor groups on the proteins. The amino acids histidine, cysteine, trypto- ∗ Tel.: +46-46-2228264; Fax.: +46-46-2224713 e-mail: Bo.Mattiasson@biotek.lu.se phan 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 prop- erties (Hemdan and Porath, 1985). This concept is also gaining usefulness for applying the technique in different formats like IMA partitioning in aqueous two phase polymer system (Birkenmeier et al., 1991; Franco et al., 1997; Otto and Birkenmeier, 1993) or metal chelate affinity precipitation (Galaev and Matti- asson, 1993; Galaev et al., 1997). This makes it more feasible and cost effective when the intended applica- tions are for large-scale processes. Aqueous two-phase partitioning for the separation and fractionation of proteins, cells and cell particles is a well-established technique and is based upon the ability of aqueous soluble polymers such as dextran and poly(ethylene glycol) (PEG), to form biphasic systems (Albertsson, 1986). The selective application of this method utilizes the interaction of proteins with affinity ligands which are covalently attached to one of the phase-forming polymers, thus extracting a specific kind of protein into one of the phases while the bulk of proteins re- main in the other phase. The IMA concept is extended to phase partitioning by grafting a chelator, iminodiac- etate (IDA) onto PEG and the ligand is able to chelate transition metal ions which have affinity for the target proteins (Birkenmeier et al., 1991).