Refolding chromatography with immobilized mini-chaperones (proteinyrenaturationyfoldingyGroELyhsp60)

Mini-chaperones (e.g., a peptide consisting of residues 191-345 of GroEL) that are immobilized on agarose have very efficient chaperoning activity with several proteins that are otherwise recalcitrant to renaturation by conventional methods. We have used immobilized mini-chaperones both in column chromatography and batchwise to renature an insoluble protein from an inclusion body, to refold apparently irreversibly denatured proteins, and to recondition enzymes that have lost activity on storage. Refolding chromatography offers an efficient and simple means to renature proteins in high yield and with biological activity. The molecular chaperone GroEL, a tetradecamer of 57 kDa subunits, facilitates the folding in vitro of a number of proteins that would otherwise misfold or aggregate and precipitate. It is cylindrical, consisting of two seven-membered rings that form a large cavity (1, 2) that has been generally considered to be essential for activity (3). In vivo, and for many reactions in vitro, GroEL requires the cochaperonin GroES (7 3 10 kDa subunits) and ATP to be functional. Although there are reports that proteolytic fragments of 34 kDa and 54 kDa have weak chaperoning activity (4, 5), monomers of GroEL that are induced by mutagenesis (6), pressure, or urea are inactive (7–9). A 54-kDa fragment of GroEL is reported as having about 10% activity in chaperoning the refolding of rhodanese when the fragment is covalently attached to a resin (5). Recombinant fragments of GroEL (including the 16.7-kDa fragment GroEL(191–345) and the 21-kDa GroEL(191–376), which were expressed in Escherichia coli) have efficient chaperone activity in facilitating the refolding of cyclophilin A and rhodanese, and the unfolding of barnase (10). Here, we attach the mini-chaperones to agarose gels and find that they can be used for the efficient renaturation of proteins. MATERIALS AND METHODS Materials. The apical domain of GroEL [GroEL(191–376)] and the ‘‘core’’ of the apical domain, GroEL(191–345), were cloned and expressed in E. coli as fusion proteins containing a 17-residue N-terminal histidine tail (10). Glucosamine 6-phosphate deaminase was expressed in and purified from E. coli K12, and assayed as described (11). Indole 3-glycerol phosphate synthase (IGPS) mutants were expressed in E. coli (M.M.A., J. Blackburn, and A.R.F., unpublished data). Cyclophilin A was purchased from Sigma or was a gift from G. Fischer and assayed as described (12). Firefly luciferase was purchased from Sigma and assayed (13). Immobilization of Mini-Chaperones. The fragments were immobilized by two methods. Attachment to Ni-NTA resin. The Ni-NTA resin (Qiagen, Chatsworth, CA) is a chelating adsorbant composed of a high surface concentration of nitrilotriacetic acid (NTA) ligand attached to Sepharose CL-6B. Proteins containing six-residue His affinity tags, located at either the amino or carboxyl terminus of the protein, bind to the Ni-NTA resin with high affinity (Kd 5 10213 M, pH 7.8). The stability of the 6 3 HisyNi-NTA interaction is unaffected by strong denaturants such as 6 M guanidine hydrochloride or 8 M urea, or the presence of low levels of 2-mercaptoethanol (1–10 mM). Ni-NTA resin (3.5 ml) was equilibrated with refolding buffer (0.1 M potassium phosphate at pH 7.8, containing 5 mM 2-mercaptoethanol). The mini-chaperone was added to saturation of the affinity gel (21 mg of protein per 3.5 ml of gel) and incubated at room temperature for 30 min with gentle mixing. The gel was packed in a column suitable for fast protein liquid chromatography (FPLC) (5 3 100 mm; Pharmacia) and thoroughly washed with refolding buffer. Attachment to CNBr-activated Sepharose 4B. To minimize the steric effects and preserve the structure of the binding site in the apical domain, the binding capacity of the gel was reduced by controlled hydrolysis of the activated gel before coupling. Freeze-dried powder (300 mg) was suspended in 50 mM NaHCO3 at pH 8.3, washed with the same buffer, and reswollen on a sintered glass filter (G3), then suspended in the buffer and mixed in an end-over-end shaker for 4 hr at room temperature. The mini-chaperone, dissolved in the coupling buffer (0.1 M NaHCO3, pH 8.3y0.5 M NaCl), was added to the gel suspension (10 mg protein per ml gel) and mixed in an end-over-end shaker for 6 hr at room temperature. It was then washed with the coupling buffer. The remaining active groups were blocked by adding 2.5 M ethanolamine at pH 8 and shaking for 4 hr at room temperature. Uncoupled minichaperone was removed by washing with five cycles of alternately high and low pH buffer solution (0.1MTriszHCl, pH 7.8, containing 0.5 M NaCl) followed by acetate buffer (0.1 M, pH 4, plus 0.5 M NaCl). The gel was finally washed with 5–10 gel volumes of refolding buffer. Batchwise Renaturation. A suspension of 200 ml of gel (wet sedimented volume, either bound via the Ni-NTA linkage or covalently linked to mini-chaperone via CNBr activation) was mixed with refolding buffer to give a volume of 990 ml. Cyclophilin A (10 ml; 100 mM stock solution in refolding buffer 1 8 M urea 5 1 nmol of cyclophilin A) was added to the suspension and mixed in an up-down mixer for 30 min at room temperature. The gel suspension was centrifuged to separate the supernatant ('800 ml). The gel pellet was washed in empty The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. Copyright q 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA 0027-8424y97y943576-3$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations: IGPS, indole 3-glycerol phosphate synthase; NTA, nitrilotriacetic acid. *Permanent address: Departamento de Bioquimica, Facultad de Medicina, Universidad Nacional de México 04510, Mexico, DF. †Present address: Martin-Luther-Universität Halle-Wittenberg, Institut für Biochemie, Kurt-Mothes Strasse, 3D-06120 Halle (Saale), Germany. ‡Present address: Institut für Molekularbiologie, Biophysik, Eidgenössiche Technische Hochschule Honggerberg (HPM G5), CH-8093 Zürich, Switzerland.