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Link:  Pharm/Biotech Resources

Title:  Monoclonal antibody to the stabilizer peptide of the P64K antigen of Neisseria meningitidis

United States Patent:  6,921,809

Issued:  July 26, 2005

Inventors:  Cano; Carlos Antonio Durate (Habana, CU); Nieto; Enrique Gerardo Guillen (Habana, CU); Acosta; Anabel Alvarez (Habana, CU); Munoz; Luis Emilio Carpio (Sancti Spiritus, CU); Vazquez; Diogenes Quintana (Pinar del Rio, CU); Rodriquez; Carmen Elena Gomez (Habana, CU); Rodriquez; Recardo De La Caridid Siva (Habana, CU); Galvez; Consuelo Nazabal (Habana, CU); Angulo; Maria De Jesus Leal (Habana, CU); Dunn; Alejandro Miguel Martin (Habana, CU)

Assignee:  Centro de Ingenieria Genetica y Biotechnologia (CU)

Appl. No.:  612925

Filed:  July 10, 2000


The present invention related to biotechnology and genetic engineering, particularly the expression of proteins of viral origin in microorganisms through their fusion by applying recombinant DNA technology to bacterial peptides. The present invention provides an effcient process for the expression in Escherichia coli of heterlogous proteins as fusion peptides with a view to obtaining them with a high degree of purity, in commercially useful amounts, and in an appropriate form for their inclusion in vaccine preparations. What is essentially used is a stabilizing sequence derived from the first 47 amino acids of the antigen P64k of Neisseria meningitides B:4:P1.15. In particular, use is made of a recombinant plasmid containing said sequence, under the control of the tryptophane promoter of E. coli and of the terminator of the transcription of the phage T4, including restrictions sites which provide for the cloning in phase of DNA fragments coding for polypeptides of interest.

Description of the Invention


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.


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, Mass., 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 (Cysteine) 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 antigen presenting cells (Del Val, M., Schlicht, H., Ruppert, T., et al, Cell, 66, 1145-1153, 1991), being able to even affect seriously the immunogenicity of the candidate by the phenomenon of specific-epitope suppression (Etlinger, H., Immunol. Today, 13, 52-55, 1992).

As a result of the aforementioned phenomenon, in some cases, small fragments that still stabilize the expression have been tried to be defined. For example, the German patent application No. 35 41 856 A1 (Hoechst AG) reports the possibility of using a stabilizer peptide conformed by at least the first 95 amino acids of the N-terminus of the human protein Interleukine (IL-2) to obtain fusion proteins in an insoluble form synthesized in E. coli. Similarly in the European Patent Applications No. 0 416 673 A2 and No. 229 998 from the same company, a stabilizer peptide consistent in the first 58 or 38 amino acids of said protein, is used. In the European patent No. 416 673 B1, the first 58 amino acids of the IL-2 are also used, and a similar strategy is followed, with this purpose, in the case of use of N-terminal fragments of the human seroalbumin (European patent application No. EP 0423641 A1 920212); the activator peptide III of the connective tissue (WO 90136647 A1 901115) and fragments of the human kallikrein (EP 0381433 A1 900808). These inventions give solution to the previous problem, but the fusion polypeptides obtained can not be included in vaccine preparations for use in humans, due to the possibility of induction of autoimmune diseases for the presence in them of homologous or identical sequences to human proteins.

The alternative of using stabilizer polypeptides of bacterial origin-and therefore, without cross reactivity with antigens of human origin-for intracellular expression, has also been explored with success. One of the most used proteins with this end has been the β-galactosidase of E. coli (Itakura, K. et al., Science, 198, 1056-1063, 1977) or portions of it (German patent application No. EP 0235754 A2 870909, of the company Hoechst AG). The principal disadvantage of this system is the great size of this protein which provokes that the desired peptide only represents a small portion of the total hybrid protein (Flores, N. et al., Appl. Microbiol. Biotechnol. 25, 267-271, 1986; Goeddel, D. V. et al., P.N.A.S. USA, 76, 106-110, 1979). Similar problems are presented with the use of the C fragment of the tetanus toxoid and the exotoxin of Pseudomonas sp. (International Patent Application PCT WO 9403615 A1 940217 and European Patent Application EP 0369316 A2 900523). An expression variant that is very promising is the use of fusions with the thioredoxin of E. coli (PCT Patent application No. WO 9402502 A1 940203), that uses the property of being liberated from the cell by osmotic stress (el Yasgoubi, A., Kohiyama, M., Richarme, G., J. Bacteriol., 176, 7074-7078, 1994) to facilitate the purification. However, this outline is not functional for the obtainment of inclusion bodies, since the same are not freed through this procedure.

Many of these problems have been solved with the design of modular fusion proteins. In these, the stabilizer peptide is separated from the protein of interest by a spacer that permits the independent folding of both, and whose amino acid sequence makes it susceptible to the attack of specific endopeptidases. If there is a ligand that recognizes the chosen stabilizer, it is possible to purify the fusion polypeptide by affinity chromatography and finally separate it from the stabilizer through the treatment with different proteases (Cress, D., Shultz, J. and Breitlow, S., Promega Notes, 42, 2-7, 1993). An additional advantage is the possibility of exploiting this molecular interaction for the follow-up of intermediate steps of the purification, without the need of antibodies for each protein to express. A well-known example of that is the use of the affinity of histidine (Hys) with some metals like nickel (Ni) and zinc (Zn) in systems composed of a stabilizer with 6 His in tandem and an affinity matrix of nickel chelates, according to what is described in the PCT Patent application No. WO 9115589 A1 911017 of The Upjohn Co. In spite of all this, this kind of expression system does not function in all the cases, since, among other reasons, the protein of interest can have restriction sites for the chosen protease, or be folded so that the spacer is available to the solvent (Uhlen, M. and Moks, T., Meth. Enzymol. 185, 129-143,1990; Cress, D., Shultz, J. and Breitlow, S., Promega Notes, 42, 2-7, 1993), to interfere with the binding between the stabilizer and the affinity matrix (New England Biolabs, The NEB Transcript, 3, 1, 1991), or simply to require, for its purification, conditions that affect its biological activity. For these reasons it is desirable to have different variants, since each protein to express can represent a particular case. With this purpose, stabilizer peptides have been developed based on the maltose binding protein of E. coli (MalE), which have affinity for the amylose resins (European Patent Application EP 0426787 A1 910515); in the chloramphenicol acetyl transferase enzymes (European Patent Application No. EP 0131363 A1 850116) or in the glutathione-S-transferase (European Patent Application No. EP 0293249 A1 88130, of the Amrad Corp., Ltd.) obtainable with matrixes of immobilized substrate; in the protein A of Staphylococcus aureus, according to the patent application PCT WO 9109946 A1 910711; and in the 12.5 kDa subunit of the transcarboxylase complex of Proprionibacterium shermanii, which is biotinylated in vivo and permits the purification based on the affinity of the biotin to avidin (Cress, D., Shultz, J. and Breitlow, S., Promega Notes, 42, 2-7,1993; patent applications No. EP 0472658 A1 920304 or WO 9014431 A1 901129).

Of particular interest is the method described in the European Patent Application EP 0472658 A1 920304 or WO 9014431 A1 901129, developed by Biotechnology Research and Development Corporation, along with the University of Illinois, USA. In this application an expression system is described that uses the lipoic acid binding domain of the dihydrolipoamida acetyltransferese (EC, also known as the E2 subunit of the pyrovate dehydrogenase complex of E. coli. This domain is modified postranslationally in vivo by the addition of a lipoic acid molecule to the nitrogen of one of its lysines (Guest, J. R., Angler, J. S. and Russell, G. C., Ann. N.Y. Acad. Sci., 573, 76-99, 1989), which is exploited for the purification and identification of fused proteins through the use of an antibody that recognizes only lipoylate domains.

This method, however, has a number of drawbacks. First of all, it is known that the over expression of proteins containing binding domains to the lipoic acid exceeds the capacity of cellular lipoylation, producing as a consequence no lipoylates domains (Miles, J. S. and Guest, J. R., Biochem. J., 245, 869-874, 1987; Ali, S. T. and Guest, J. R., Biochem. J., 271, 139-145) or octanoilates (Ali, S. T., Moir, A. J., Ashton, P. R. et al. Mol. Microbiol., 4, 943-950, 1990; Dardel, F., Packman, L. C. and Perham, R. N., FEBS Lett. 264, 206-210, 1990), which can reduce the yield during purification by immunoaffinity. In second place, there are a group of diseases of a supposed autoimmune origin which have as a common factor the presence of antibodies that recognize specifically the lipoic acid in the context of these domains. Among them are primary biliary cirrhosis, a chronic disease characterized by the inflammation and progressive obstruction of the intrahepatic bile ducts (Tuaillon, N., Andre, C., Briand, J. P. et al., J. Immunol., 148, 445-450, 1992); and hepatitis and the hepatitis provoked by halothane, an anesthetic of wide use that derivatizes some proteins by the formation of trifluoroacetyl lysine (Gut, J., Christen, U., Frey, N. et al, Toxicology, 97, 199-224, 1995). The serum of the patients with this disease recognizes said complexes, whose molecular structure is mimicked by the lipoic acid in the context of the dihydrolipoamide acetyl transferases (Gut, J., Christen, U., Frey, N. et al., Toxicology, 97, 199-224, 1995). For this reason it is desirable to avoid the presence of the lipoic acid in such peptides if the fusion proteins that contain it constitute vaccine candidates for use in humans.


An object of the present invention is a procedure for the expression to high levels of heterologous proteins as fusion polypeptides in E. coli, which is based on the use of a stabilizer sequence derivative from the first 47 amino acids of the P64K antigen of N. meningitidis B:4:P1.15 (European Patent application No. 0 474 313 A2) that confers on them the capacity of being expressed as inclusion bodies. Said sequence, though presents homology with part of the lipoic acid binding domain of the dihydrolipoamide acetyl transferases, has been genetically manipulated to eliminate the possibility of modification for itself and presents the advantage of being lowly immunogenic. This procedure also includes the use of a monoclonal antibody that specifically recognizes the mentioned stabilizer, permitting the immunodetection of any protein fused to the same.

Particularly, in the present invention, a recombinant plasmid as an expression vector is used which carries said sequence under the control of the tryptophan promoter (ptrip) of E. coli, followed by restriction sites XbaI, EcoRV and BamHI. These permit the in frame cloning of DNA fragments encoding for polypeptides of interest. This vector also includes a terminator of the transcription of the gene 32 of bacteriophage T4 and a resistance gene to ampicillin as selection marker.

This procedure makes possible also the inclusion of the fusion polypeptide obtained in vaccine preparations destined to be used in humans; and the nature of the stabilizer peptide employed permits the generation of protective immune response against the foreign protein or the multiepitopic peptide bound to it.

A novelty of the present invention is the genetic manipulation and the use of an homologous stabilizer peptide to part of the lipoic acid binding domain of the dihydrolipoamide acetyl transferases, for the production of fusion proteins by recombinant DNA technology in E. coli. Particularly, novelties of the present invention are the use, with the previous objective, of a stabilizer peptide derivative of the first 47 amino acids of the P64K antigen of N. meningitidis B:4:P1.15 (European Patent application No. 0 474 313 A2), and a monoclonal antibody that specifically recognizes the stabilizer.

The values obtained show that the titers against the V3 regions are similar between the varying IL2-22+MEP (TAB4) and P64K-47+MEP (TAB9). Though the recognition frequency of the peptides is slightly greater for the TAB9, this difference is not meaningful statistically (p<0.05). In conclusion, the immunogenicity of the heterologous protein is affected by the stabilizer P64K-47 in a minimal way, and comparable to other expression systems currently in use.

Claim 1 of 1 Claim

1. An isolated monoclonal antibody 448/30/7 that is produced from a hybridoma deposited on Sep. 24, 2003 with the Belgian Coordinated Collections of Microorganisms, BCCM™/LMBP-COLLECTION, Department of Molecular Biology, Ghent University, Fiers-Schell-Van Montagu building, Technologiepark 927,B-9052 Zwijnaarde, Belgium, under deposit number LMBP 6047CB, wherein the monoclonal antibody that specifically recognizes for a stabilizer peptide consisting of the amino acid sequence:

MVDKRMALVELKVPDIGGHENVDIIAVEVNVGDTIAVDDTLITLDLE (SEQ. ID. NO. 15) the amino acid sequence being the first 47 amino acids of the N-terminal end of the P64K antigen of Neisseria meningitidis B:4:P1.15.

If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.



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