Chaperonin and osmolyte protein folding and related screening methods

#This invention relates to a method of in vitro protein folding. More particularly, the method employs both chaperonins and osmolytes to optimize protein folding as well as to aid in the screening for optimal folding solution conditions.

#Efficient refolding of proteins in vitro is an important problem in protein structural analysis and biotechnological manufacturing of pharmaceutical products. Because of their inherent ability to rapidly overexpress proteins to high yields, bacterial systems are the organisms of choice for protein mass production. Unfortunately, overexpression of foreign and, especially, mutant proteins often leads to the development of large intracellular aggregates or inclusion bodies (Rudolph, R and Lilie, H. (1996) FASEB J. 10, 49-56; Guise, A. D., West, S. M., and Chaudhuri, J. B. (1996) Mol. Biotechnol. 6, 53-64, the disclosures of which are incorporated herein by reference). In some cases, the proper intracellular folding of the overexpressed proteins can be enhanced by lowering the cell growth temperature, co-expressing molecular chaperones, or introducing low molecular weight additives (Kujau, M. J., Hoischen, C., Riesenberg, D., and Gumpert, J. (1998) Appl. Microbiol. Biotechnol. 49, 51-58; Tate, C. G., Whiteley, E., and Betenbaugh, M. J. (1999) J. Biol-Chem. 274, 17551-17558; Minning, S., Schmidt-Dannert, C., Schmid, R. D. (1998) J. Biotechnol. 66, 147-156, the disclosures of which are incorporated herein by reference). More often, however, investigators are forced to rely on in vitro folding methods to denature (also known as “deactivate”) and then refold (also known as “reactivate”) aggregated proteins. A number of in vitro approaches have been developed to minimize protein aggregation and enhance proper refolding. Among those are: (1) the addition of osmolytes and denaturants to refolding buffer (Tate, C. G., Whiteley, E., and Betenbaugh, M. J. (1999) J. Biol-Chem. 274, 17551-17558; Plaza-del-Pino, I. M. and Sanchez-Ruiz, J. M. (1995) Biochemistry 34, 8621-8630, Frye, K. J. and Royer, C. A. (1997) Protein. Sci. 6: 789-793, the disclosures of which are incorporated herein by reference); (2) the use of the combinations of different molecular chaperones (Thomas, J. G., Ayling, A., and Baneyx, F. (1997) Appl. Biochem. Biotechnol. 66, 197-238; Buchberger, A., Schroder, H., Hesterkamp, T., Schonfeld, H. J., and Bukau, B. (1996) J. Mol. Biol. 261, 328-233; Veinger, L., Diamant, S., Buchner, J., and Goloubinoff, P. (1998) J. Biol. Chem. 273, 11032-11037, the disclosures of which are incorporated herein by reference); (3) immobilization of folding proteins to matrices and matrix-bound chaperonins (Stempfer, G., Holl-Neugebauer, B., and Rudolph, R. (1996) Nat. Biotechnol. 14, 329-334; Altamirano, M. M., Golbik, R., Zahn, R., Buckle, A. M., and Fersht, A. R. (1997) Proc. Natl. Acad. Sci. USA 94, 3576-3578; Preston, N. S., Baker, D. J., Bottomley, S. P., and Gore, M. G. (1999) Biochim. Biophys. Acta 1426, 99-109, the disclosures of which are incorporated herein by reference); and (4) utilization of folding catalysts such as protein disulfide isomerase and peptidyl-prolyl cis-trans isomerase (Altamirano, M. M., Garcia, C., Possani, L. D., and Fersht, A. R. (1999) Nat. Biotechnol. 17, 187-191, the disclosure of which is incorporated herein by reference). Unfortunately, because of the diversity of the protein folding mechanisms, there is no universal procedure for protein folding and folding conditions have to be optimized for each specific protein of interest. Therefore, there is always a need for new and more versatile folding techniques. This invention involves a novel protein folding procedure that combines the use of the GroE chaperonins and cellular osmolytes.

#Because of its ability to bind many different protein folding intermediates, it was thought that the bacterial GroE chaperonin system could provide a general method to refold misfolded proteins. Chaperonin GroEL is a tetradecamer of identical 57 kDa subunits that possesses two large hydrophobic sites capable of binding to transient hydrophobic protein folding intermediates. The hydrophobic binding site undergoes the multiple cycles of exposure and burial driven by the ATP binding and hydrolysis and the co-chaperonin GroES binding and dissociation. Accordingly, the protein folding intermediates can undergo multiple rounds of binding to and release from the GroEL until they achieve the correctly folded state (for review, see Fenton, W. A. and Horwich, A. L. (1997) Protein Sci. 6, 743-760, the disclosure of which is incorporated herein by reference). Besides simple prevention of non-productive aggregation, chaperonins may also influence the conformation of the folding intermediates, actively diverting them to a productive folding pathway (Fedorov, A. N. and Baldwin, T. O. (1997) J. Mol. Biol. 268, 712-723; Shtilerman, M., Lorimer, G., and Englander, S. W. (1999) Science 284, 822-825, the disclosures of which are incorporated herein by reference). However, despite the general nature of chaperonin-protein interactions, there are many proteins that, for reasons that are currently unknown, cannot fold correctly from the bacterial chaperonin system.

#FIG. 1A and FIG. 1B show the kinetics of spontaneous and chaperonin-dependent renaturation of wild type and mutant GS.

#FIGS. 2A and 2B compare the assembly time of wild type GS and GSΔ468 in the presence of chaperonins. The set of arrows in FIG. 2 indicates the GS monomers, dimers, tetramers, and higher multimers produced by time-dependent association of native GS from the chaperonin.

#FIG. 3 shows the chaperonin-dependent renaturation of wild type and mutant GS in the presence of glycerol.

#As used herein, “protein” is defined as a polypeptide or polypeptide chain having a native or “active” form with a known biological function and a denatured form which does not exhibit the biological function of the native form.

#As used herein, “chaperonin” is defined as any protein complex that binds to an unfolded polypeptide to facilitate the folding of said polypeptide to its biologically active state either independently or with the assistance of other elements. This definition specifically includes but is not limited to chaperonin systems from bacteria and bacteriophages, including mesophiles and thermophilic chaperoning. Similarly, as used herein, chaperonin includes but is not limited to chaperonins in any native or modified state, for example, single ring chaperoning, glutaldehyde cross-linked chaperonins or other chemically modified chaperoning.

#As used herein, “unfolded”, “denatured” and “inactive” are defined interchangeably to mean the characteristic of polypeptides which are no longer biologically active due, at lease in part, to not being in their native shape. As such, the terms include partially folded proteins, chemically unfolded proteins, thermally denatured proteins, pressure unfolded proteins, and oxidatively damaged proteins.