Solid-Phase Refolding of Cyclodextrin Glycosyltransferase Adsorbed on Cation-Exchange Resin Dae-Hyuk Kweon, † Dae-Hee Lee, Nam-Soo Han, ‡ and Jin-Ho Seo* Department of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Korea, School of Bioresource Sciences, Andong National University, Andong, Kyungbuk 760-749, Korea, and Department of Food Science and Technology, Chungbuk National University, Chungju, Chungbuk 361-763, Korea Expression with a fusion partner is now a popular scheme to produce a protein of interest because it provides a generic tool for expression and purification. In our previous study, a strong polycationic tail has been harnessed for an efficient purification scheme. Here, the same polycation tail attached to a protein of interest is shown to hold versatility for a solid-phase refolding method that utilizes a charged adsorbent as a supporting material. Cyclodextrin glycosyltransferase (CGTase) fused with 10 lysine residues at the C-terminus (CGTK10ase) retains the ability to bind to a cation exchanger even in a urea-denatured state. When the denatured and adsorbed CGTK10ase is induced to refold, the bound CGTK10ase aggregates little even at a g/L range. The renatured CGTK10ase can also be simply recovered from the solid support by adding high concentration of NaCl. The CGTK10ase refolded on a solid support retains specific enzyme activity virtually identical to that of the native CGTK10ase. Several factors that are important in improving the refolding efficiency are explored. Experimental results indicate that nonspecific electrostatic interactions between the charge of the ion exchanger and the local charge of CGTase other than the polycationic tag should be reduced to obtain higher refolding yield. The solid- phase refolding method utilizing a strong polycationic tag resulted in a remarkable increase in the refolding performance. Taken together with the previous report in which a series of polycations were explored for efficient purification, expression of a target protein fused with a strong polycation provides a straightforward protein preparation scheme. Introduction In the post-genome period, there is a strong demand for a rapid preparation of pharmaceutical and industrial proteins with native conformation. The common and popular way is to express a target protein using recom- binant microbial systems including Escherichia coli. Formation of inclusion bodies, often observed upon overexpression of heterologous proteins in several host systems (1), offers several advantages for the production of proteins because this process is generally not very sensitive to proteolytic breakdown. In addition, under appropriate conditions, foreign proteins deposited in the inclusion bodies amount to about 50% or more of the total cellular proteins (2). However, refolding of inclusion bodies is not a straightforward process. Main challenges lie in the tedious steps of unfolding and refolding for the recovery of biological activity. The efficiency of a refolding process depends on the competition between correct folding and aggregation. To minimize aggregation, refolding is usually performed at low protein concentrations in a range of 10-100 mg/L (3). Refolding conditions must be carefully selected on the basis of temperature, pH, additives, ionic strength, and concentrations of the denaturant and the protein itself (4). Even under an optimized condition, the yield of renaturation is often relatively low, necessitating large process volumes for the preparation of the native protein. Therefore, research activities often have been focused on the protection of intermolecular aggregation, either by changing the processes for the removal of the denatur- ants (5-7) or by adding aggregation-protecting molecules (8, 9). A strategy employing the immobilization of target proteins on a solid phase has been proposed to circumvent aggregation of unfolded proteins or folding intermediates because proteins attached to an insoluble carrier may avoid intermolecular aggregation. Preparative protein refolding in the solid phase requires that the proteins be reversibly attached to a solid support such as Ni resin or ion-exchange resin in the presence of a denaturant. Refolding of the proteins reversibly adsorbed to an ion exchanger has been performed for several proteins (10). These proteins were absorbed on cation or anion exchang- ers through their own charges. Ni-NTA agarose has been also employed using the His 6 fusion tag (11). However, solid-phase refolding using the own charge of the protein is often accompanied by the problem of the promiscuous electrostatic interactions between the arbitrarily distrib- uted local charge of the denatured protein and the * To whom correspondence should be addressed. Tel: +82-2- 880-4855. Fax: +82-2-873-5095. E-mail: jhseo94@snu.ac.kr. † Andong National University. ‡ Chungbuk National University. 277 Biotechnol. Prog. 2004, 20, 277-283 10.1021/bp0341895 CCC: $27.50 © 2004 American Chemical Society and American Institute of Chemical Engineers Published on Web 10/22/2003