title: Solid-phase refolding of cyclodextrin glycosyltransferase adsorbed on cation-exchange resin.
abstract: 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.
authors.affiliation: Department of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Korea.
authors.forename: Dae-Hyuk
authors.initials: DH
authors.lastname: Kweon
authors.forename: Dae-Hee
authors.initials: DH
authors.lastname: Lee
authors.forename: Nam-Soo
authors.initials: NS
authors.lastname: Han
authors.forename: Jin-Ho
authors.initials: JH
authors.lastname: Seo
mesh_terms: D000327:Adsorption; D002411:Cation Exchange Resins / Q000737:chemistry*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004793:Enzyme Reactivators / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005964:Glucosyltransferases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D011489:Protein Denaturation; D015202:Protein Engineering / Q000379:methods; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D002411:Cation Exchange Resins; D004793:Enzyme Reactivators; D011993:Recombinant Fusion Proteins; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase; D008239:Lysine