1. Field of the Invention
The invention relates to the field of recombinant protein production.
2. Related Art
Proteins for industrial applications, e.g. for use as biopharmaceuticals or fine chemicals, are either obtained by extraction and purification from a natural source, such as a plant or animal tissue or microorganisms, or by means of recombinant DNA technology.
To produce a recombinant protein, the cDNA encoding the protein of interest is inserted into an expression vector and the recombinant vector is transformed into host cells, which are grown to overexpress the protein. The host cells may be selected from microorganisms such as bacteria, yeast or fungi, or from animal or plant cells.
Overexpression of a protein is a complex event. To obtain the correct conformation, the protein, already in its native state, is associated with so-called “folding helper proteins” and enzymes. The folding helper proteins, also termed “chaperones” or “minichaperones”, interact in a complex way so that the protein regains its native conformation after passing through various intermediate states. Some of the intermediate states may be quite stable. Enzymes involved in protein maturation either catalyze the rapid formation of disulfide bridges (Horwich, A. L., et al., Trends Biotechnol. 8:126–131 (1990); Noiva, R., Protein Expr Purif. 5:1–13 (1994)), the isomerization of prolyl-peptide linkages (Schonbrunner, E. R., et al., Proc. Natl. Acad. Sci. (USA) 89:4510–4513 (1992); Lilie, H., et al., Protein Sci. 2:1490–1496 (1993); Jager, M., et al., FEBS Lett. 418:106–110 (1997); Yang, H. P., et al., Biochim Biophys Acta 1338:147–150 (1997)) or more complex modifications, such as the truncation of the protein, side chain modifications or modifications of the N- and C-terminus. When a protein is efficiently overexpressed, the production of the nascent peptide chain occurs faster than the folding of the protein. For some proteins, an intermediate state may also form aggregates (in the following, the term “intermediate” forms also encompasses aggregate forms).
Overall, aggregate formation occurs much faster than the complete folding of a protein (Kane, J. F. and D. L., H., TIBTECH 6:95–100 (1988); Buchner, J. and Rudolph, R., Current Opinion Biotechnology 2:532–538 (1991)).
In expression systems, in which such conditions are present, the protein is deposited in the cells in a paracrystalline form, so-called “inclusion bodies”, also termed “refractile bodies”.
Since the protein, when present in the form of insoluble inclusion bodies, is shielded from enzymatic attack, such as proteolysis, and cannot interfere with the physiology of the cells. Recombinant DNA technology has taken advantage of this aberrant way of protein secretion, e.g. for the production of the proteins that are toxic for the cells.
To obtain a protein from host cells, in which it is accumulated in a denatured form, i.e. a conformational state without biological activity, various steps have to be taken to obtain the protein in its correctly refolded form. For example, bacterial cells carrying inclusion bodies are disintegrated, the inclusion bodies harvested by centrifugation and then dissolved in a buffer containing a chaotropic agent. The denatured protein is then transferred into an environment that favours the recovery of its native conformation. Before adopting its native state, the protein undergoes a transition through various semi-stable intermediates. Since intermediates have highly exposed hydrophobic domains, which are prone to associate, they tend to form aggregates. In principle, refolding may be considered as a race against aggregate formation, which usually follows second order reaction kinetics, while refolding of the protein follows first order reaction kinetics (Buchner, J. and Rudolph, R., Current Opinion Biotechnology 2:532–538 (1991)).
With the currently available methods, refolding of proteins is achieved by diluting the protein in a refolding buffer in a batch or continuous mode (Halenbeck, R., et al., Bio/technology 7:710–715 (1989); Kiefhaber, T., et al., Biotechnology (NY) 9:825–829 (1991); Lilie, H., et al., Curr. Opin. Biotechnol. 9:497–501 (1998); Clark, E. D., Curr Opin Biotechnol. 12:202–207 (2001); Yoshi, H., et al., J. Chem. Eng. (Japan) 34:211–215 (2001)). In these methods, batchwise dilution results in highly diluted protein solutions and therefore large process volumina, which often is the bottleneck in industrial processes.
In another approach the folding pathway is simulated in vivo by adding chaperons and/or minichaperons, and/or enzymes that catalyze certain steps in the folding pathway (Noiva, R., Protein Expr Purif. 5:1–13 (1994); Buchner, J., et al., Biotechnology (N.Y.) 10:682–685 (1992); Carlson, J. D. and Yarmush, M. L., Biotechnology (N.Y.) 10:86–91 (1992); Guise, A. D. and Chaudhuri, J. B., Biotechnol. Prog. 14:343–346 (1998); Kohler, R. J., et al., Biotechnol. Prog. 16:671–675 (2000); Shimizu, H., et al., Biotechnol. Prog. 16:248–253 (2000)). Complex refolding reactor systems comprising series of tanks have been designed to improve the refolding reaction (Katoh, S. and Katoh, Y., 2000 35:1119–1124 (2000)).
In another approach, the helper proteins and enzymes are immobilized to a solid phase. Then the protein solution is passed over a so-called Packed Bed containing the immobilized helper proteins and/or helper enzymes, thus being folded into its native conformation (Phadtare, S., et al., Biochim Biophys Acta 1208:189–192 (1994); Altamirano, M. M., et al., Proc Natl. Acad. Sci. USA 94:3576–3578 (1997); Altamirano, M. M., et al., Nat Biotechnol, 17:187–191 (1999); Preston, N. S., et al., Biochim Biophys Acta 1426:99–109 (1999)). Since the folding helper proteins and enzymes must be present in a stoichiometric ratio, this process requires almost the same amount of helper proteins, which in turn have to be produced by recombinant DNA technology, as the finally obtained product. In addition, to improve folding, the helper proteins are usually fused to the protein of interest, which requires further processing of the fusion protein. For these reasons, this strategy is very cost intensive.
Since a certain protein fraction is lost in the form of aggregates, refolding of the protein in free solution or in the matrix-assisted process is not efficient enough to transfer the denatured protein into the folded form in a quantitative way.
A protein can be refolded from its denatured conformation to the correctly folded conformation by transferring it into an environment that favors the change to the native conformation. During this rearrangement, the protein passes through several intermediate conformational states, which are prone to form aggregates. Depending on the individual protein and on the environmental conditions, the aggregates may precipitate. Independent of whether the aggregates remain soluble or whether they precipitate, this process leads to dramatic losses in the yield of correctly folded protein. In general, the folding of a protein to its native conformation follows first order reaction kinetics, while the formation of aggregates from intermediates follows second or higher order reaction kinetics.
It was an object of the invention to provide an efficient method for refolding a protein from a denatured state, which overcomes the shortcomings of the currently used methods and which can be operated without using helper proteins.
The solution of the problem underlying the invention is based on the consideration that the chromatographic separation process may be improved by running it continuously. In addition, it was hypothesized that recycling the intermediate forms of the protein may further allow both to improve the yield of a recombinant protein and to work at high protein concentrations, which would significantly reduce the process volume.
USPTO Backgrounds
USPTO Backgrounds