ELSEVIER Biochimica et Biophysica Acta 1252 (1995) 69-78 BB Biochi~ic~a et Biophysica A~ta Isolation and biochemical characterization of highly purified Escherichia coli molecular chaperone Cpn60 (GroEL) by affinity chromatography and urea-induced monomerization Andreas Blennow a,*, Brian P. Surin b, Hanno Ehring c, Neil F. McLennan d, Michael D. Spangfort e Department of Biochemistry, Chemical Center, University of Lund, S-221 O0 Lund, Sweden b CSIRO Division of Plant Industry, Canberra ACT 2601, Australia c Department of Radiation Sciences, University of Uppsala, S-751 21 Uppsala, Sweden J Institute of Cell and Molecular Biology, University of Edinburgh, Mayfield Road, Edinburgh EH9 3JR, UK e ALK Laboratories, Protein Chemistry Department, DK-2970 HCrsholm, Denmark Received 1 November 1994; revised 29 March 1995; accepted 9 May 1995 Abstract Isolated Escherichia coli molecular chaperone Cpn60 (GroEL) has been further purified from tightly bound substrate polypeptides by two different procedures: (i) group-specific affinity chromatography by using the triazine dye Procion yellow HE-3G as affinity ligand, and (ii) urea-induced monomerization and subsequent chromatography. Procion yellow binds specifically to aromatic amino-acid side chains present in the majority of proteins, but has no affinity to GroEL because of its low content of aromatic residues. Some GroEL-bound polypeptides are buried within the aqueous cavity of the GroEL oligomer, whereas others are exposed on its surface and available for affinity-ligand interactions and the complex is thereby retarded on Procion yellow columns. Pure substrate-free GroEL was obtained after ion-exchange chromatography of GroEL monomers followed by reassembly of the purified monomers into functional GroEL oligomers. The final preparation contained no substrate polypeptides bound to GroEL as judged by electrophoretic analysis and lack of tryptophan fluorescence. GroEL preparations also displayed two equally strong bands on native electrophoresis suggesting the presence of two conformer~. Monomers of GroEL showed heterogeneity with respect to isoelectric point and molecular mass when analysed by MALDI-MS and electrophoresis under native and denaturing conditions respectively. By use of MALDI-MS, highly accurate molecular masses of wild-t)pe and a truncated form of GroEL were determined and verified, by comparison with their respective gene sequences. Keywords: Chaperone Cpn60; Affinity chromatography; GroEL; Urea-induced monomerization; (E. coli) 1. Introduction The Escherichia coli protein GroEL is a molecular chaperone which plays an essential role in the folding of newly synthesized polypeptides and in the refolding of partly denatured polypeptides [1]. GroEL and its homo- Abbreviations: CBB, Coomassie brilliant blue; DTT, dithiothreitol; EDTA, ethylenediaminetetraa,=etic acid; IEF, isoelectric focusing; MALDI, matrix associated laser desorption/ionization mass spectrome- try; PMSF, phenylmethylsulfonylfluoride; SDS-PAGE, sodium dodecyl- sulfate polyacrylamide gel electrophoresis. * Corresponding author. Fax: +46 46 104534. 0167-4838/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 01 67-483 8(95)0011 1-5 logues are present in prokaryotes, plastids and mito- chondria [2]. Under in vitro conditions, GroEL has been shown to form complexes with denatured polypeptides which in some cases are highly stable [1]. Bound polypep- tide substrate is released from GroEL upon addition of MgATP, K + and the molecular co-chaperone GroES, con- comitant with the correct refolding of the substrate [3]. In some cases, GroES is not needed and correct folding is obtained in the presence of MgATP and K+[ 1]. GroEL is a large oligomeric protein consisting of 14 protomers of 57 kDa each, kept together mainly by hy- drophobic interactions [4]. Electron-microscopy of nega- tively stained GroEL particles [5] and X-ray analysis of