Use of Protein Folding Reagents

Paul T. Wingfield1

1 Protein Expression Laboratory, National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), NIH, Bethesda, Maryland
Publication Name:  Current Protocols in Protein Science
Unit Number:  Appendix 3A
DOI:  10.1002/0471140864.psa03as84
Online Posting Date:  April, 2016
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


The reagents and methods for purification and use of the most commonly used denaturants, guanidine hydrochloride (guanidine‐HCl) and urea, are described. Other protein denaturants and reagents used to fold proteins are briefly mentioned. Sulfhydryl reagents (reducing agents) and “oxido‐shuffling” (or oxidative regeneration) systems are also described. © 2016 by John Wiley & Sons, Inc.

Keywords: protein folding; guanidine hydrochloride; urea; protein denaturants; sulfhydryl reagents; oxidative regeneration

PDF or HTML at Wiley Online Library

Table of Contents

  • Denaturants
  • Sulfhydryl Reagents and Oxido‐Shuffling Systems
  • Tables
PDF or HTML at Wiley Online Library


PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
  Adamczyk, J., Bal, W., and Kręzel, A. 2015. Coordination properties of dithiobutylamine (DTBA), a newly introduced protein disulfide reducing agent. Inorg. Chem. 54:596‐606. doi: 10.1021/ic5025026.
  Bajaj, K., Chakshusmathi, G., Bachhawat‐Sikder, K., Surolia, A., and Varadarajan, R. 2004. Thermodynamic characterization of monomeric and dimeric forms of CcdB (controller of cell division or death B protein). Biochem. J. 380:409‐417. doi: 10.1042/bj20031528.
  Baneyx, F. and Palumbo, L. 2003. Improving heterogenous protein folding via molecular chaperones and folding co‐expression. Meth. Mol. Biol. 205:171‐197.
  Bondos, S.E. and Bicknell, A. 2003. Detection and prevention of protein aggregation before, during, and after purification. Anal. Biochem. 316:223‐231. doi: 10.1016/S0003‐2697(03)00059‐9.
  Bull, H.R., Breese, K., Ferguson, G.L., and Swenson, G.L. 1964. The pH of urea solutions. Arch. Biochem. Biophys. 104:297‐304. doi: 10.1016/S0003‐9861(64)80017‐5.
  Burgess, R.R. 2009. Refolding solubilized inclusion body proteins methods. Enzymology 463:259‐279. doi: 10.1016/S0076‐6879(09)63017‐2.
  Burns, J.A., Burns, J.C., Butler, J.C., Moran, J., and Whitesides, G.M. 1991. Selective reduction of disulfides by tris(2‐carboxyethy1)phosphine. J. Org. Chem. 56:2648‐2650. doi: 10.1021/jo00008a014.
  Clarke, D.T. 2012. Circular dichroism in protein folding studies. Curr. Protoc. Protein Sci. 70:8.3.1‐8.3.17.
  Cleland, W.W. 1964. Dithiothreitol, a new protective reagent for SH groups. Biochemistry 3:480‐482. doi: 10.1021/bi00892a002.
  Creighton, T.E. 1984. Disulfide bond formation in proteins. Methods Enzymol. 107:305‐329. doi: 10.1016/0076‐6879(84)07021‐X.
  England, J.L. and Haran, G. 2011. Role of solvation effects in protein denaturation: From thermodynamics to single molecules and back. Annu. Rev. Phys. Chem. 62:257‐277. doi: 10.1146/annurev‐physchem‐032210‐103531.
  Grimsley, G.R., Trevino, S.R., Thurlkill, R.L., and Scholtz, J.M. 2013. Determining the conformational stability of a protein from urea and thermal unfolding curves. Curr. Protoc. Protein Sci. 71:28.4.1‐28.4.14.
  Hidaka, Y. 2014. Overview of the regulation of disulfide bond formation in peptide and protein folding. Curr. Protoc. Protein Sci. 76:28.6.1‐28.6.6.
  Hun, J.C. and Hun, G.Y. 1994. A procedure for quantitative determination of Tris (2 carboxyethyl) phosphine, an odorless reducing agent more stable than dithiothreitol. Anal. Biochem. 220:5‐10. doi: 10.1006/abio.1994.1290.
  Hwang, C., Sinskey, A.J., and Lodish, H.F. 1992. Oxidized redox state of glutathione in the endoplasmic reticulum. Science 257:1496‐1502. doi: 10.1126/science.1523409.
  Jocelyn, P.C. 1987. Chemical reduction of disulfides. Methods Enzymol. 143:246‐264. doi: 10.1016/0076‐6879(87)43048‐6.
  Kawahara, K. and Tanford, C. 1966. Viscosity and density of aqueous solutions of urea and guanidine hydrochloride. J. Biol. Chem. 241:3228‐3232.
  Kim, Y.E., Hipp, M.S., Bracher, A., Hayer‐Hartl, M., and Hartl, F.U. 2013. Molecular chaperone functions in protein folding and proteostasis. Annu. Rev. Biochem. 82:323‐355. doi: 10.1146/annurev‐biochem‐060208‐092442.
  Lim, W.K., Rosgen, J., and Englander, S.W. 2009. Urea, not guanidinium, destabilizes proteins by forming hydrogen bonds to the peptide bond. Proc. Natl. Acad. Sci. 106:2595‐2600. doi: 10.1073/pnas.0812588106.
  Lukesh, III, J.C., Palte, M.J., and Raines, R.T. 2012. A potent, versatile disulfide‐reducing agent from aspartic acid. J. Am. Chem. Soc. 134:4057‐4059. doi: 10.1021/ja211931f.
  Mamathambika, B.S. and Bardwell, J.C. 2008. Disulfide‐linked protein folding pathways. Annu. Rev. Cell Dev. Biol. 24:211‐235. doi: 10.1146/annurev.cellbio.24.110707.175333.
  Means, G.M. and Feeney, R.E. 1971. Chemical Modification of Proteins. Holden‐Day, San Francisco.
  Nozaki, Y. 1972. The preparation of guanidine hydrochloride. Methods Enzymol. 26:43‐50. doi: 10.1016/S0076‐6879(72)26005‐0.
  Nozaki, Y. and Tanford, C. 1967. Acid‐base titrations in concentrated guanidine hydrochloride. Dissociation constants of the guanidinium ion and some amino acids. J. Amer. Chem. Soc. 89:736‐742. doi: 10.1021/ja00980a002.
  Pace, C.N. 1986. Determination and analysis of urea and guanidine hydrochloride denaturation curves. Methods Enzymol. 131:266‐280. doi: 10.1016/0076‐6879(86)31045‐0.
  Pain, R.H. 1995. Overview of protein folding. Curr. Protoc. Protein Sci. 00:6.4.1‐6.4.7.
  Pain, R. 2005. Determining the CD spectrum of a protein. Curr. Protoc Protein Sci. 38:7.6.1‐7.6.24.
  Prakash, V., Loucheux, C., Scheufele, S., Gorbunoff, M.J., and Timasheff, S.N. 1981. Interactions of proteins with solvent components in 8 M urea. Arch. Biochem. Biophys. 210:455‐464. doi: 10.1016/0003‐9861(81)90209‐5.
  Street, T.O., Bolen, D.W., and Rose, G,D. 2006. A molecular mechanism for osmolyte‐induced protein stability. Proc. Natl. Acad. Sci. 103:13997‐14002. doi: 10.1073/pnas.0606236103.
  Tisher, A., Lilie, H., Rainer, R., and Lange 2010. l‐Arginine increases the solubility of folded and unfolded recombinant plasminogen activator rPA. Prot. Sci. 19:1783‐1795. doi: 10.1002/pro.465.
  Tsumoto, K., Umetsu, M., Kumagai, I, Philo, J.S., and Arakawa, T. 2008. Role of arginine in protein refolding, solubilization and purification. Biotech. Progress 20:1301‐1308. doi: 10.1021/bp0498793.
  Tu, B.P. and Weissman, J.S. 2004. Oxidative protein folding in eukaryotes mechanisms and consequences. J. Cell Biol. 164:341‐346. doi: 10.1083/jcb.200311055.
  Vallejo, L.F. and Rinas, U. 2004. Strategies for the recovery of active proteins through refolding of bacterial inclusion body proteins. Microbial. Cell Factories 3:11. doi:10.1186/1475‐2859‐3‐11.
  Wetlaufer, D.B. 1984. Nonenzymatic formation and isomerization of protein disulfides. Methods Enzymol. 107:301‐304. doi: 10.1016/0076‐6879(84)07020‐8.
  Williams, A. and Frasca, V. 1999. Ion‐exchange chromatography. Curr. Protoc. Protein Sci. 15:8.2.1‐8.2.30.
  Wingfield, P.T., Palmer, I., and Liang, S.‐M. 2014. Folding and purification of insoluble (inclusion body) proteins from Escherichia coli. Curr. Protoc. Protein Sci. 78:6.5.1‐6.5.30. doi: 10.1002/0471140864.ps0605s78
  Wetlaufer, D.B., Branca, P.A., and Chen, G.X. 1987. The oxidative folding of proteins by disulfide plus thiol does not correlate with redox potential. Protein Eng. 1:141‐146. doi: 10.1093/protein/1.2.141.
Internet Resources‐
  Bio‐Rad Bulletin 1825. Sample preparation: A guide to methods and applications. BioRad Laboratories, Hercules, Calif.
PDF or HTML at Wiley Online Library