Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Fusion protein or fusion polypeptide
Patent
1997-09-16
2000-11-14
Housel, James C.
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Fusion protein or fusion polypeptide
4241851, 4241901, 4242081, 4242501, 4242491, 4241841, 530350, 530825, 530820, A61K 3900, A61K 3902, A61K 3921, A61K 39095
Patent
active
061466352
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL SECTOR
The present invention is related to the field of the Biotechnology and the genetic engineering, particularly to the expression of heterologous proteins in microbial hosts through their fusion to bacteria peptides, using the technology of the recombinant DNA.
PREVIOUS ART
The usefulness of the technology of the recombinant DNA to produce proteins of any origin in E. coli has been extensively demonstrated. For this, an important amount of vectors have been developed, although new variants are necessary due to the fact that, frequently each gene to clone and to express represents an individual case (Denhardt, D. T. and Colasanti, J.; Vectors ed., Butterworths, Stoneham, MA, Biotechnology 10, 179-203, 1988 and Lukacsovich, T. et al., Journal of Biotechnology, 13, 243-250, 1990).
The intracellular synthesis has been the most used strategy for the obtainment of heterologous polypeptides in E. coli, due to the high expression levels reachable (Goeddel, D. V, Methods Enzymol., 185, 3-7, 1990). However, factors such as the sensitivity to proteases of the host or toxicity of the expressed protein can reduce significantly said levels, independently of the use of regulatory sequences of high efficiency (Lee, C. A. and Saier, M. H., J. Bacteriol., 153, 685-692, 1983; Gwyn, G. W., Membrane Protein Expression Systems: A User's Guide, Portland Press, London, UK, 29-82, 1992). The cloning of nucleotide sequences encoding for proteins of interest in suitable vectors, in frame with sequences of nucleic acid that encode stable polypeptides in the host cell, gives rise to the expression of hybrid products in the cytoplasm, known as fusion proteins (Marston, F. A. O., Biochem. J. 240, 1-12, 1986). Such polypeptides are generally less sensitive to proteolytic degradation by the host or less toxic due to the formation of inclusion bodies, which results in higher expression levels to those obtained without the use of the stabilizer peptide (Itakura, K. et al., Science, 198, 1056-1063, 1977). In addition, this kind of expression facilitates and cheapens the initial steps of the purification if different methods for the subsequent renaturation of the recombinant product are available (Fischer, B., Sumner, I. and Goodenough, P., Biotechnol. Bioeng., 41, 3-13, 1993).
The inclusion bodies are insoluble protein aggregates that appear as electrodense bodies in the cytosol during the expression of many recombinant proteins in E. coli (Rinas, U. and Bailey, J., Appl. Microbiol. Biotechnol., 37, 609-614, 1992). They are the result of the interaction between polypeptides partially folded, whose aggregation is thermodynamically favored due to the exposition, within them, of hydrophobic residues to the solvent (Kiefhaber, T., Rudolph, R. et al., Biotechnology, 9, 825-829, 1991). The slow folding in the bacterial cytosol of many eukaryotic proteins, due to the abundance of disulfide bridges-forming amino acids (Cysteino) or beta-turn-forming amino acids (Proline) has stimulated the abundant use of them as stabilizer peptides. Examples of the former are the use, with this purpose, of polypeptides with binding activity to antibodies, coming from the globulin of the fat of the human milk (HMFG), according to the international patent application PCT No. WO 9207939 A2 920514; from constant regions of the immunoglobulins, as described in the European patent application No. EP 0464533 A1 920108; from the human angiogenin (European patent application No. EP 0423641 A2 910424), of the growth hormone (EP 0429586 A1 910605), the glutatione-S-tranferase (WO 8809372 A1 881201) and of the swine adenylate quinase (EP 0423641 A2 910424 and EP 0412526 A2 910213).
However, the use of stabilizer polypeptides that constitute a significant part of the fusion protein has some disadvantages if the former is a vaccine candidate, since the presence of the foreign sequences can alter the natural order of the B and T cell epitopes (Denton, G., Hudecz, F., Kajtar, J. et al., Peptide Research, 7, 258-264, 1994) or the processing of the same by the anti
REFERENCES:
patent: 5286484 (1994-02-01), Rodriguez et al.
Niebla et al. In: Neisseria 94, Proceedings of the Ninth International Pathogenic Neisseria Conference, (Ed) Evans et al. Winchester, England, Sep. 26-30, 1994.
Guillen et al. Biotechnol. Apl. 12: 72, 1995.
Guillen et al. Biotechnol. Apl. 13: 271-275, 1996.
Acosta Anabel Alvarez
Angulo Maria De Jesus Leal
Cano Carlos Antonio Durate
de la Caridid Siva Rodriguez Recardo
Dunn Alejandro Miguel Martin
Centro de Ingenieria Genetica y Biotecnologia
Devi S.
Housel James C.
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