MX2007006330A - Production of proteins - Google Patents

Abstract

A method for forming a fusion protein that is expressed as a recombinant protein body-like assembly in host eukaryotic cells and organisms other than higher plants as host systems is disclosed. More particularly, peptides and proteins are fused to protein sequences that mediate the induction of recombinant protein body-like assembly (RPBLA) formation, are stably expressed and accumulated in these host cells after transformation with an appropriate vector. Methods for preparing the fusion protein are also disclosed.

Description

"PROTEIN PRODUCTION" Technical Field The present invention relates to the production of peptides and recombinant proteins in eukaryotic cells and organisms other than higher plants as host systems. More specifically, the peptides and proteins are fused into protein sequences that mediate the induction of the formation of recombinant protein bodies (ESCPR) assemblies, and are expressed and accumulated with stability in these host systems after being transformed with a vector suitable. Prior Art The production of recombinant proteins for therapeutic, nutritional or industrial uses has enjoyed great success during the past decade. It has been shown that different eukaryotic cells and organisms are capable of producing active therapeutic compounds based on proteins. Unfortunately, the high costs that are often derived from low production levels of recombinant proteins and / or protein isolation and purification procedures can invalidate their industrial application. Active research is carried out to improve both production levels and purification procedures from different approaches. A new technology has been developed based on the fusion of the domain of a seed storage protein of a plant with the protein of interest (WO 2004/003207) to increase the stability and accumulation of recombinant proteins in higher plants. These storage proteins are specific to plant seeds, in which they accumulate with stability in protein bodies (Galili et al., 1993, Trends Cell Biol 3: 437-442). The storage proteins are inserted into the lumen of the endoplasmic reticulum (ER) by means of a signal peptide, and assembled in the endoplasmic reticulum by developing specific organelles called protein bodies derived from ER (CP-RE), or in the vacuoles of protein storage (VAP) (Okita and Rogers 1996 Annu. Rev. Plant Physiol Mol. Biol. 47: 327-50; Herman and Larkins 1999 Plant Cell 1 1: 601 -613; Sanderfoot and Raikel 1999 Plant Cell 1 1: 629-642). It has also been described that recombinant storage proteins gather in protein-like organelles (CP) in different host systems of plants, such as Xenopus oocytes and yeast. The expression of cereal prolamins (the most abundant cereal storage proteins) has been described in Xenopus oocytes after injection of the corresponding mRNAs. This system has been used as a model to study the targeting properties of these storage proteins (Simón et al., 1990, Plant Cell 2: 941-950, Altschuler et al., 1993, Plant Cell 5: 443- 450; Torrent et al., 1994, Plant 192: 512-518) and to test the possibility of modifying the a-zein of 19 kDa, a prolamin of corn, by introducing the essential amino acids lysine and tryptophan in their sequence, without altering its stability (Wallace et al., 1988, Science 240: 662-664). Zeins, the complex group of corn prolamines, have also been produced in yeast for various purposes. Coraggio et al., 1988, Eur J Cell Biol 47: 165-172, expressed native a-zeins and modified yeast to study the target determinants of this protein. Kim et al., 2002, Plant Cell 14: 655-672, studied the possible interactions of a, ß,? and d-zeins that lead to the formation of protein bodies. To answer this question, they transformed the yeast cells with cDNAs that encode these proteins. In addition, the authors constructed zein-GFP fusion proteins to determine the subcellular localization of zein proteins in the yeast cells. The yeast cells, then, were used as a model expression system to study the properties of zein. It should be noted that Kim et al., 2002, Plant Cell 14: 655-672, concluded that yeast is not a good model for studying zein interactions because zeins, by themselves, did not accumulate well in the leaven transformed. Yeast cells were also used as a model to study the mechanisms that control the transport and deposition of protein bodies from wheat storage proteins called gliadins (Rosenberg et al., 1993, Plant Physiol 102: 61-69).

In the present document we demonstrate that the fusion of a sequence of the protein that mediates the induction of assemblies similar to recombinant protein bodies (ESCPR), such as, for example, prolamins or prolamin domains with a peptide or protein of interest (target) it also mediates the accumulation of said ESCPR in cells of organisms such as fungi (including yeast), algae and animals. Interestingly, these fusion proteins accumulate with stability in animal cells, within structures in organelles similar to protein bodies.

COMPENDIUM OF THE INVENTION The present invention provides a system and method for producing a fusion protein containing a protein body-inducing sequence (CPIS) and a peptide or protein of interest (in this document they are often called "polypeptides" collectively) in cells eukaryotes that do not belong to higher plants, such as animal cells, fungi and algae, as well as in cultured cells of animals, fungi and algae in which the fusion proteins containing the peptide or protein of interest accumulate with stability in the form of assemblies similar to recombinant protein bodies (ESCPR). The CPIS are capable of mediating the induction of ESCPR formation and the entry of proteins and / or accumulation in these organelles, such as, for example, the natural and modified sequences of the storage protein sequences with a peptide or protein. interest (targets). The present invention provides, inter alia, a method for producing a product of interest in the form of a fusion protein, in eukaryotic cells that do not belong to higher plants as a host system that has been transformed with a nucleic acid sequence comprising a portion of nucleic acid encoding the CPIS and a portion of nucleic acid encoding a polypeptide product of interest. In a particular embodiment, the nucleic acid sequence used for the transformation comprises (i) a nucleic acid sequence encoding a CPIS and (ii) a nucleic acid sequence comprising the nucleotide sequence encoding a product of interest. In one embodiment, the 3 'end of the nucleic acid sequence (i) is linked to the 5' end of said nucleic acid sequence (ii). In another embodiment, the 5 'end of the nucleic acid sequence (i) is linked to the 3' end of the nucleic acid sequence (ii). Thus, the CPIS sequence may be at the N-terminal or C-terminal end of the fusion protein. In another specific embodiment, the nucleic acid sequence used for transformation comprises, in addition to the nucleic acid sequences above or mentioned (i) and (ii), a nucleic acid sequence comprising the nucleotide sequence encoding the sequence amino acid spacer The amino acid spacer sequence can be a cleavable, or non-cleavable, amino acid sequence by enzymatic or chemical means. In a particular embodiment, the nucleic acid sequence (iii) is placed between the nucleic acid sequences (i) and (ii), for example, the 3 'end of the nucleic acid sequence (iii) is linked to the 5' end of said nucleic acid sequence (ii). In another embodiment, the 5 'end of said nucleic acid sequence (iii) is linked to the 3' end of the nucleic acid sequence (i). Also, in a specific embodiment, the nucleic acid sequence used for the transformation encodes a specifically cuttable sequence and is as defined in the patent application WO 2004003207, granted together with the present application. Furthermore, in another embodiment, the nucleic acid is in accordance with the patent application WO 2004003207, in which the nucleic acid sequence encoding the amino acid sequence that is specifically cleavable by enzymatic or chemical means is absent. In a further embodiment, the fusion proteins can be a direct fusion between the CPIS and the peptide or protein of interest. In another embodiment, the method of the invention further comprises the isolation and purification of the fusion protein. In another further embodiment, the protein of interest is fused to a natural or modified storage protein, such as for example the natural or modified prolamines or the prolamin domains. Examples of proteins of interest are any protein with therapeutic, nutritional, biocontrol or industrial use. Some illustrative proteins and peptides are, for example, hormones such as calcitonin, growth hormone and the like, antibodies such as monoclonal antibodies and fragments thereof, antigens such as those which are useful for vaccines against the human immunodeficiency virus (HIV).; surface or nuclear proteins of hepatitis B, gastroenteritis, coronavirus and the like, protease, inhibitors, antibiotics, collagen, human lactoferrin, cytokines, industrial enzymes, such as hydrolases, glucosidases, oxido-red uctases, etc.

BRIEF DESCRIPTION OF THE FIGURES In the figures that form an integral part of this description: Figure 1 is a photograph of an SDS / PAGE analysis showing the accumulation of fusion proteins, including calcitonin (Ct), human growth hormone (hGH) and factor of epidermal growth (EGF) as individual interest proteins linked to the RX3 protein body-inducing sequence derived from gamma zein in cells of transfected mammals. The fusion proteins RX3-Ct, RX3-hGH and RX3-EGF accumulated in the cultured cells of transfected mammals 293T, CHO and Cosi are shown. Equivalent amounts of transfected mammalian cells were extracted at 44 hours post-transfection, and the corresponding soluble total proteins were analyzed with electrophoresis and Western blot technique using the anti-zein gamma antibody. Also included are schematic representations of the constructs encoding the fusion proteins RX3-Ct, RX3-hGH and RX3-EGF. "c" = cells; "m" = medium. Molecular weight markers (in kDa) are indicated on the left. Figure 2 is a six panel photograph (A-F) showing the location of the fusion proteins in the ESCPR within transfected cells. Confocal microscopy was used to show Cosí cells expressing RX3-CT (Fig. 2A), CHO cells expressing RX3-Ct (Fig. 2B) and RX3-EGF (Fig. 2C), and 293T cells coexpressing RX3-Ct and the protein marker DsRed2-RE (Fig. 2D, Fig. 2E and Fig. 2F). The fusion proteins derived from RX3 were immunolocalized with anti-zein gamma serum in structures similar to protein bodies (Figs 2A-2D) and in the endoplasmic reticulum (see arrow in Fig. 2A). Fig. 2E shows the colored ER with the fluorescent red protein marker DsRed2-RE. Fig. 2F shows an overlay of Figures 2D and 2E and represents the colocalization of RX3-Q with DsRed2 in the RE and in the CPLS. The figures embedded in Figures 2A and 2C show very enlarged images of the CPLS. Bars: 1 micrometer. Figure 3 on two panels (A and B) illustrates the accumulation of fusion proteins in transformed yeast cells. Figure 3A shows the accumulation of fusion proteins RX-EGF (Slot C1 17) and RX-hGH (Slot c1 18) in transformed Saccharomyces. Equivalent amounts of total cell protein extracts and immunoblot media were analyzed using specific antibodies. Cells transformed with the plasmid pYX243 without insert were used as control (C). The lower part of each panel contains a schematic representation of the constructs encoding the fusion proteins RX3-hGH and RX3-EGF. Figure 3B illustrates the accumulation of hGH and RX3-hGH containing fusion proteins in Pichia pastoris using two different signal peptides. Equivalent amounts of total cell protein extracts and transformed media were analyzed with immunoblot using specific antibodies. C1 and C2 indicate cells transformed, respectively, with plasmids pPIC9 and pPIC3.5K used as control. Schematic representations of the c135 and c121 constructs encoding the RX3-hGH fusion protein and the c136 construct encoding hGH are shown in the lower part of the panels. Molecular weight markers (in kDa) are indicated to the left of the figures, "and" = yeast cells; "m" = medium; "SPg" = zein gamma signal peptide; "RX3" = N-terminal sequence of proline-rich gamma zein, "EGF" = epidermal growth factor; "hGH" = human growth hormone; "Afprepro" = peptide factor alpha prepro. The present invention offers several benefits and advantages. One benefit is that their use allows the relatively simple and rapid expression of a desired recombinant protein in a chosen eukaryotic cell that does not belong to higher plants. An advantage of the invention is that it provides a source of recombinant protein easily obtainable and purifiable due to expression in RBPLA. Other additional benefits and advantages will be apparent to those skilled in the art from the following description.DETAILED DESCRIPTION OF THE INVENTION The contemplated recombinant proteins are fusion proteins that form assemblages similar to recombinant protein bodies (ESCPR) in the host cells in which they are expressed. The formation of ESCPR is induced with storage protein domains that form high density deposits within cells. These dense deposits can accumulate cytosol, in an organelle of the endomembrane system, mitochondria, plastids or can be secreted. Assemblies similar to recombinant protein bodies have a predetermined density that can differ between different fusion proteins, but which is known for a specific fusion protein being prepared. This predetermined density of the ESCPR is usually higher than that of almost all the endogenous proteins of host cells present in the homogenate, and is usually from about 1.1 to 1.35 g / ml. The high density of the new ESCPR is due to the general ability of recombinant fusion proteins to form multimers and accumulate. The contemplated RBPLAs are expressed in eukaryotes that do not belong to higher plants and are usually characterized by their densities as described above. When expressed in animal cells, ESCPRs usually have a spherical shape, with diameters of approximately 1 micrometer (μ) and have a surrounding membrane. These fusion proteins comprise two polypeptide sequences linked directly or indirectly by a peptide bond, wherein a sequence is a protein body-inducing sequence (CPIS) linked to a polypeptide product (e.g., peptide or protein) of interest (target ). CPIS are sequences of protein or peptide amino acids that mediate the induction of ESCPR formation and the entry and / or accumulation of proteins in organelles. A CPIS and the host cell are preferably of different biological phyla. Thus, the CPIS is normally of a higher plant, a spermatophyte, while the host cell is a eukaryotic different from a spermatophyte and can be an animal cell, such as for example mammalian or insect cells, a fungus or yeast, or a cell of seaweed, all of which are of different edges than the spermatophytes. Exemplary CPIS examples, but not restrictives, are storage proteins or modified storage proteins, such as prolamines or modified prolamines, prolamin domains or modified prolamin domains. Prolamines are analyzed in Shewry et al., 2002 J. Exp. Bot. 53 (370): 947-958. Preferred CPISs are those of prolamin compounds such as zein gamma, alpha zein or rice prolamin mentioned below. Gamma zein, a corn storage protein whose DNA and amino acid residue sequences are shown below, is one of four corn prolamines and accounts for 10 to 15 percent of the total protein found in the corn. endosperm of corn. Like other cereal prolamins, alpha- and zein gammas are biosynthesized in polysomes bound with membranes on the cytoplasmic side of the rough ER, they gather within the light and then they are sequestered in the protein bodies derived from the ER (Herman et al., 1999 Plant Cell 1: 601-613; Ludevid et al., 1984 Plant Mol. Biol. 3: 277-234; Torrent et al., 1986 Plant Mol. Biol. 7: 93-403). The gamma zein is composed of four characteristic domains: i) a signal peptide of 19 amino acids, ii) a repeating domain containing eight units of hexapeptide PPPVHL (SEQ ID NO: 1) (53 aa), iii) the ProX domain where the proline residues alternate with other amino acids (29 aa) and iv) the C-terminal hydrophobic domain rich in cysteine (111 aa). The ability of the gamma zein to constitute ESCPR derived from the ER is not restricted to the seeds. In fact, when the gamma zein gene was constitutively expressed in the Arabidopsis transgenic plants, the storage protein accumulated within the CPLS derived from the ER in the mesophilic cells of the leaves (Geli et al., 1994 Plant Cell 6: 191 1 -1922). When looking for the signal responsible for the deposition of gamma zein in the protein bodies derived from the ER (prolamins do not have the KDEL signal), it has been shown that the proline-rich N-terminal domain including the tandem repeat domain was necessary for the retention of the ER and that the C-terminal domain participated in the formation of the protein bodies. However, the mechanisms by which these domains promote the composition of protein bodies are still unknown. In the same way that protein bodies are only properly named in seeds, similar structures produced in other plant organs and in non-superior plants are generally called recombinant protein bodies (ESCPR) assemblies. Other useful prolamin-type sequences are shown by way of illustration in the following table along with their GenBank identifiers.

Other useful sequences are obtained by a BLAST search in the complete non-redundant database of GenBank including CDS translations + PDB + SwissProt + PIR + PRF (excluding environmental samples) as described in Altschul et al., 1997 Nucleic Acids Res. 25 : 3389-3402 using an unknown such as those shown below: sequence of the RX3 protein (SEQ ID NO: 2), alpha-zein protein sequence (SEQ ID NO: 3), and prolamin protein sequence of the rice (SEQ ID NO: 4). A prolamin modified by way of illustration comprises (a) a sequence of signal peptides, (b) a sequence of one or more copies of the repeated hexapeptide domain PPPVHL (SEQ ID NO: 1) of the gamma zein protein, the entire domain contains eight hexapeptide units; and (c) a sequence of all or part of the ProX domain of the gamma zein. Some illustrative specific modified prolamines are the polypeptides identified below as R3, RX3 and P4 whose DNA sequences and amino acid residues are also shown below. Particularly preferred prolamines are gamma zein and its component parts as disclosed in published application WO2004003207, rP13 rice protein and 22 kDa alpha zein of corn and its N-terminal fragment. The DNA sequences and amino acid residues of the gamma, rice and zein alpha proteins are shown below: - Zein gamma (27 KD) SEQ ID NO: 5 (DNA sequence) and SEQ ID NO: 6 (protein sequence).

- RX3 SEQ ID NO: 7 (DNA sequence) and SEQ ID NO: 8 (protein sequence) - R3 SEQ ID NO: 9 (DNA sequence) and SEQ ID NO: 10 (protein sequence) - P4 SEQ ID NO: 1 1 (DNA sequence) and SEQ ID NO: 12 (protein sequence) - X10 SEQ ID NO: 13 (DNA sequence) and SEQ ID NO: 14 (protein sequence) - rP13 (13 kD rice prolamin homologous to the clone - (GenBank AB016504) Sha et al., 1996 Biosci Biotechnol Biochem 60 (2): 335-337; Wen et al., 1993 Plant Physiol. 101 (3 ): 1 115-1 1 16; Kawagoe et al., 2005 Plant Cell 17 (4): 1 141-1 153; Mullins et al., 2004 J. Agrie. Food Chem. 52 (8): 2242-2246; Mitsukawa et al., 1999 Biosci Biotechnol Biochem 63 (11): 1851-1858) SEQ ID NO: 15 (protein sequence) and SEQ ID NO: 16 (DNA sequence). - 22aZt [N-terminal fragment of the 22kD corn zein zein - (GenBank V01475) Kim et al., 2002 Plant Cell 14 (3): 655-672; Woo et al., 2001 Plant Cell 13 (10): 2297-2317; Matsushima et al., 1997 Biochim. Biophys. Acta 1339 (1): 14-22; Thompson et al., 1992 Plant Mol. Biol. 18 (4): 827-833]. SEQ ID NO: 17 (protein sequence) and SEQ ID NO: 18 (DNA sequence) Examples of proteins of interest include any protein having therapeutic, nutritional, biocontrol or industrial uses, such as for example monoclonal antibodies (mAb such as IgC, IgM, IgA, etc.) and fragments thereof, antigens for vaccines ( human immunodeficiency virus, HIV, pre-surface antigens, surface and core of hepatitis B, coronaviruses of gastroenteritis, etc.), hormones (calcitonin, growth hormone, etc.), protease inhibitors, antibiotics, collagen , human lactoferrin, cytokines, industrial enzymes (hydrolases, glucosidases, oxido-reductases, and the like).

Exemplary sequences of DNA and amino acid residues for proteins of interest are, for example, Salmon Calcitonin, BAC57417 [SEQ ID NO: 19 (protein sequence) and SEQ ID NO: 20 (DNA sequence)]; hEGF, construction based on AAF85790 without the signal peptide, [SEQ ID NO: 21 (protein sequence) and SEQ ID NO: 22 (DNA sequence)]; and hGH, construction based on P01241 without the signal peptide [SEQ ID NO: 23 (protein sequence), SEQ ID NO: 24 (DNA sequence)]. In another embodiment, the recombinant fusion protein comprises in addition to the CPIS and product sequences of interest, a spacer sequence of amino acids. The spacing sequence of amino acids can be a cuttable sequence, or non-cuttable, by enzymatic or chemical means. In a specific embodiment, the amino acid spacer sequence is placed between the CPIS and the product of interest. An illustrative amino acid sequence is cuttable with a protease such as an enterokinase, endoprotease Arg-C, endoprotease Glu-C, endoprotease Lys-C, Factor Xa and the like. Alternatively, an amino acid sequence that is specifically cleavable with a chemical reagent, such as, for example, cyanogen bromide that cuts into the methionine residues, is encoded. In a further embodiment, the nucleic acid sequence used for transformation is as disclosed in co-pending patent application WO 2004003207. Furthermore, in another embodiment, the nucleic acid sequence is as disclosed in patent application WO 2004003207, but the nucleic acid sequence encoding the cleavable amino acid sequence is absent. In a preferred embodiment, the fusion proteins are prepared according to a method comprising the transformation of a host system of eukaryotic cells different from higher plants, such as an animal, culture of animal cells, fungi / yeast, insects or algae with a sequence of nucleic acid (DNA or RNA) comprising (i) a first nucleic acid encoding a CPIS that is operatively linked in the same reading frame to (ii) a second nucleic acid sequence comprising the nucleotide sequence encoding a product of polypeptide interest, i.e., the nucleic acid sequence encoding the CPIS is chemically linked (peptide linkage) to the sequence encoding the polypeptide of interest so that both polypeptides are expressed from their own reading patterns. The host cell is maintained for a period of time and under culture conditions suitable for expression of the fusion protein and composition of the fusion protein expressed in recombinant protein bodies-like assemblies (ESCPR). After expression, the resulting fusion protein accumulates in the transformed host system in the form of assembly similar to high protein recombinant protein bodies similar to protein bodies. The fusion protein can then be recovered from the host cells or the host cells containing the fusion protein can be used as desired, for example for an animal feed containing an added nutrient or supplement. The fusion protein can be isolated as part of the ESCPR or free of ESCPR. The culture conditions suitable for expression of the fusion protein are typically different for each type of host cell. However, these conditions are known to those skilled in the art and are easily determined. Similarly, the duration of maintenance may differ according to the host cells and according to the amount of fusion protein to be prepared. Again, these conditions are well known and can be easily determined in specific situations. In addition, specific growing conditions can be obtained from the documents cited in this document. In one embodiment, the 3 'end of the first nucleic acid sequence (i) is linked to the 5' end of the second nucleic acid sequence (i). In another embodiment, the 5 'end of the first nucleic acid sequence (i) is linked to the 3' end of the second nucleic acid sequence (ii). In another embodiment, the CPIS comprises a storage protein or a modified storage protein, a fragment or a modified fragment thereof or a newly designed proline-rich sequence. In another specific embodiment, a fusion protein is prepared according to a method comprising transforming the host cell system, for example an animal, animal cell culture, fungi / yeast or algae, with a nucleic acid sequence comprising, in addition to the aforementioned nucleic acid sequences (i) and (ii), a nucleic acid sequence (ii) in the pattern encoding the amino acid spacer sequence. The spacing amino acid sequence may be a cuttable or non-cutting sequence, by enzymatic or chemical means, as explained above. In a specific embodiment, the nucleic acid sequence (iii) is placed between said nucleic acid sequences (i) and (ii), for example, the 3 'end of the third nucleic acid sequence (iii) is linked to the extreme 5 'of the second nucleic acid sequence (ii). In another embodiment, the 5 'end of the third nucleic acid sequence (iii) is linked to the 3"end of the second nucleic acid sequence (ii).

A sequence (segment) of nucleic acid encoding a fusion protein molecule previously described or a complement of said coding sequence is also contemplated herein. Such a nucleic acid segment is present in isolated and purified form in some preferred embodiments. In living organisms, the sequence of amino acid residues of a protein or polypeptide is directly related by genetic code to the deoxyribonucleic acid (DNA) sequence of the gene encoding the protein. Thus, by means of the well-known degeneracy of the genetic code, DNA sequences and their corresponding RNA sequences (nucleic acids) can be prepared as desired which encode the same amino acid residue sequences of fusion proteins, but which are quite different of a genetic sequence described above to prevent the two sequences from hybridizing under highly restrictive conditions, although they do hybridize under moderate restrictive conditions. Highly restrictive conditions can be defined as those comprising hybridization at a temperature of about 50 ° -55 ° C in 6XSSC and a final wash at a temperature of 68 ° C in 1-3XSSC. Moderate stringent conditions comprise hybridization at a temperature of about 50 ° C to about 65 ° C in 0.2 or 0.3 M NaCl, followed by washing at about 50 ° C to about 55 ° C in 0.2X SSC, 0.1% SDS (sodium dodecyl sulfate). A nucleic sequence (DNA sequence or RNA sequence) that (1) codes for itself, or its complement encodes, a fusion protein containing a protein-producing sequence (CPIS) and a polypeptide of interest, as well it is contemplated in the present. As is well known, a nucleic acid sequence such as the contemplated nucleic acid sequence is expressed when it is operably linked to a suitable promoter in a suitable expression system as described elsewhere herein. Different hosts often have preferences for a specific codon to be used to encode a specific amino acid residue. Such codon preferences are well known and a DNA sequence encoding a desired fusion protein sequence can be altered, using for example in vitro mutagenesis, so that codons preferred by the host are used for a specific host in the that the fusion protein must be expressed. Also contemplated in this invention is a recombinant nucleic acid molecule such as a DNA molecule, comprising a vector containing one or more regulatory sequences (control elements) as a suitable promoter to drive the expression of the gene in an organism of eukaryotic host cells compatible operably linked to an exogenous segment of nucleic acid (e.g., a segment or DNA sequence) that defines a gene encoding a contemplated fusion protein, as described above. More specifically, a recombinant DNA molecule comprising a vector comprising a promoter to drive expression of the fusion protein in cells of the host organism operably linked to a DNA segment defining a gene encoding an inducing sequence is also contemplated. of protein bodies (CPIS) linked to a polypeptide of interest. Said recombinant DNA molecule, after suitable transfection and expression in a eukaryotic host cell, produces a fusion protein contemplated in the form of ESCPR. As is well known in the art, as long as the required nucleic acid sequence (eg, DNA) is present including the start and end signals, additional base pairs may be present at either end of the DNA segment and that segment may be used to express the protein. This, of course, presupposes the absence in the segment of an operably linked DNA sequence that represses the expression, expressing an additional product that consumes the fusion protein that is desired to express, expresses a product that consumes a desired reaction product produced by said desired fusion protein, or otherwise interferes with the expression of the gene of the DNA segment. Thus, as long as the DNA segment is free of said interfering DNA sequences, a DNA segment of the invention can have from about 500 to about 15,000 base pairs in length. The maximum size of a recombinant DNA molecule, especially an expression vector, is determined primarily by the convenience and size of the vector that a host cell can accept, given that all the minimum DNA sequences required for replication and expression , when desired, are present. The minimum sizes of vectors are well known. Said long sequences of non-5 DNA are preferred, but can be used. A DNA segment encoding a fusion protein described above can be synthesized with chemical techniques, such as for example the phosphotriester method of Matteucci et al. (1981) J. Am. Chem. Soc, 103: 3185. Of course, by chemical synthesizing the coding sequence, any desired modifications can be made simply by substitution of the appropriate bases by those encoding the native amino acid residue sequence. However, DNA segments are preferred, among them the sequences specifically described herein. The DNA segments containing a gene encoding the fusion protein are preferably obtained from recombinant DNA molecules (plasmid vectors) containing said gene. A vector that directs the expression of a fusion protein gene in a host cell is referred to herein as an "expression vector". An expression vector contains expression control elements, including the promoter. The gene encoding the fusion protein is operably linked to the expression vector to allow the promoter sequence to direct the RNA polymerase linkage and the expression of the gene encoding the fusion protein. To express the gene encoding the polypeptides, promoters that are inducible, viral, synthetic and constitutive are described as described by Poszkowski et al. 25 (1989) EMBO J., 3: 2719 and Odell et al. (1985) Nature, 313: 810, as well as those that are regulated in time, regulated in space and regulated spatiotemporally as described in Chua et al. (1989) Science, 244: 174-181. The expression vectors compatible with eukaryotic cells, such as those that are compatible with yeast cells or those compatible with cells of mammals, algae or insects and the like, are contemplated in the present. Such expression vectors can also be used to form the recombinant DNA molecules of the present invention. Vectors for use in yeasts such as S. cerevisiae or Pichia pastoris may be episomal or integrating, as is well known. Expression vectors of eukaryotic cells are well known in the art and there are several commercial suppliers. Typically, said vectors contain one or more convenient restriction sites for the insertion of the desired DNA segment and promoter sequences. Optionally, said vectors contain a specific selectable marker for use in eukaryotic cells. Examples of promoters for use in S. cerevisiae are the phosphoglycerol kinase (PGK) promoter for S. cerevisiae and the GAL 10 and GAL 1 divergent promoters, while the alcohol oxidase (AOX1) gene is a useful promoter for the Pichia pastoris An illustrative expression of a fusion protein in S. cerevisiae and Pichia pastoris is shown below. The production of a fusion protein by recombinant DNA expression in mammalian cells is illustrated hereinafter using a recombinant DNA vector expressing the fusion protein gene in Chinese hamster ovary (CHO) host cells, cells Hosts of the Cosí and human 293T. This is accomplished using procedures well known in the art and are described in more detail in Sambrook et al., Molecular Cloninq: A Laboratorv Manual, 2nd ed., Cold Spring Harbor Laboratories (1989). An insect cell system can also be used to express a contemplated fusion protein. For example, in one such system the nuclear polyhedrosis virus of Autographa californica (AcNPV) or baculovirus is used as an expression vector of foreign genes in Spodoptera frugiperda cells or Trichoplusia larvae. The sequences encoding a fusion protein can be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under the control of the polyhedrin promoter. Successful insertion of a fusion protein sequence renders the polyhedrin gene inactive and produces a recombinant virus lacking a protein coat. The recombinant viruses can then be used to infect, for example, S. frugiperda cells or Trichoplusia larvae in which the fusion protein can be expressed. E. Engelhard et al. (1994) Proc. Nati Acad. Sci., USA, 91: 3224-3227; and V. Luckow, Insect Cell Expression Technology, pp. 183-218, in Protein Enqineerinq: Principies and Practice, J.L. Cleland et al. eds., Wiley-Liss, Inc, 1996). Heterologous genes placed under the control of the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcNPV) are often expressed at high levels during the late stages of infection. Recombinant baculoviruses containing the fusion protein gene are constructed using the baculovirus transporter vector system (Luckow et al (1993) J. Virol., 67: 4566-4579], sold commercially as the expression system of baculovirus Bac-to-Bac (Life Technologies) Stocks of recombinant viruses are prepared and the expression of the recombinant protein is monitored with normal protocols (O'Reilly et al., Baculovirus Expression Vectors: A Laboratory Manual, WH Freeman and Company , New York, 1992; and King et al., The Baculovirus Expression System: A Laboratory Guide, Chapman & Hall, London, 1992). The choice of which expression vector and which promoter is operably linked to a gene encoding fusion protein depends directly on the desired functional properties, for example the location and time of expression of proteins, and the host cell to be transformed. These are well-known limitations inherent in the technique of building recombinant DNA molecules. However, a useful vector for practicing the present invention can direct the replication, and preferably also the expression (for an expression vector) of the gene of the fusion protein included in the DNA segment to which it is operatively linked. The expressed ESCPR and its fusion proteins can be obtained from the host cells expressing them by common means used in biochemical or biological recovery. As the ESCPR are dense with respect to other proteins present in the host cells, the ESCPR are especially sptible to being collected by centrifugation of a cellular homogenate. The fusion proteins can be obtained from the ESCPR collected by dissolving the surrounding membrane in a buffer solution containing a reducing agent such as 2-mercaptoethanol.

Without further elaboration, it is believed that a person skilled in the art can, using the preceding description and the examples detailed below, use the present invention to its fullest extent. The following preferred specific embodiments should, therefore, be interpreted merely as illustrative and not in any way limiting the remainder of the description.

Example 1: Accumulation of fusion proteins in cells of transfected mammals Synthetic genes corresponding to mature calcitonin (Ct) and EGF sequences as well as the cDNA encoding the hGH sequence were fused to the N-terminal gamma zein sequence which encodes RX3 (WO2004003207) and were introduced into the vector pcDNA3.1 (Invitrogen) to obtain the constructs p3.1 RXCt, p3.1 RXEGF and p3.1 RXhGH. These constructs encoding the fusion proteins RX3-Ct, RX3-EGF and RX3-hGH were introduced into mammalian 293T, Cosi and CHO cells by the lipofectamine-based transfection procedure (Invitrogen). 293T and Cosi cells transfected with plasmid pECFP-N1 (Clontech) containing the genetic sequence of an improved and modified fluorescent cyan GFP were used as controls. The accumulation of fusion proteins in the transiently transfected cells was analyzed with Western blot, using antibodies against the gamma zein. After 44 hours of transfection, the total soluble proteins were extracted with buffer solution A (100 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM EDTA, 0.5% SDS, Triton X-100 0.5 %, 2-mercaptoethanol 2% and protease inhibitors). The aliquots of cell incubation media were precipitated and stored at -20 ° C. Proteins extracted from equivalent amounts of transfected cells were separated by electrophoresis by polyacrylamide gel and transferred to nitrocellulose sheets. As can be seen from the results shown in Figure 1, the three fusion proteins analyzed RX3-Ct, RX3-EGF and RX3-hGH, accumulated very efficiently regardless of the type of cultured cells selected for expression: compare the pattern of accumulated RX3-hGH in both 293T and CHO cells and the RX3-EGF pattern in CHO and Cosí cells. The fusion proteins were observed in the protein extracts corresponding to the transfected cells (slots c) and no immunoreactive band was detected in the cell culture media (slots m). This observation suggests that the RX3 domain is capable of composing and retaining the fusion proteins in the endomembrane compartment.

These results illustrate how fusion proteins derived from RX3 are composed and accumulated in the endomembrane system in the three types of mammalian cells analyzed (human 293T cells, Cosí monkey cells and CHO hamster cells), suggesting that an efficient accumulation of a desired protein can be achieved in any mammalian cell or organism chosen by means of fusion with the RX3 domain.

Example 2: Subcellular localization of fusion proteins in transfected mammalian cells To determine whether the N-terminal gamma zein RX3 sequence was able to induce recombinant protein-like assemblies in mammalian cells, the location of the RX3 fusion proteins -Ct and RX3-EGF was analyzed with immunocytochemistry using confocal microscopy. The transfected cells were fixed for 10 minutes in 3% paraformaldehyde and after washing with phosphate buffer, they were incubated with gamma zein antiserum (1/700 solution) for 1 hour. The non-immune serum was used as a control. The primary antibodies were detected with conjugated anti-rabbit antibodies in Alexa Fluor 488 or Alexa Fluor 555 dyes (molecular probes). Micrographs of the transfected cells were obtained using a laser confocal microscope (Leica TCS SP, Heidelberg, Germany) equipped with spectrophotometers for selection of the wavelength of the emission band. The green fluorescent images were collected at a 488 nm excitation with the Argon ion laser using a defined emission window at 495-535 nm. Red fluorescent images were collected after an excitation of 543 nm with a HeNe laser and emission window of 550-600. The optical sections were 0, 5 μ ?? of thickness. Digital images and projections were recorded with confocal microscope software. Fig. 2 shows the confocal projections of cells transfected with p3.1Ct-p3.1 RXEGF. As shown in the figure, the corresponding fusion proteins, RX3-Ct and RX3EGF, were detected in the endoplasmic reticulum (ER, arrow in Figure 2A) indicating that the signal peptide of gamma zein is functional in mammalian cells in which mediates the translocation of the fusion protein to ER. The samples incubated with the non-immune serum as control did not show any significant fluorescence (not shown). It is important to note that it is surprising that the fusion proteins appear preferentially accumulated in large spots that appear surrounded by a membrane (see figure embedded in Figure 2A). These structures, which are absent in non-transfected cells, are comparable in size to the plant bodies of plants, with diameters of approximately 1 micrometer (figures embedded in A and C). This result is not only surprising because animal cells can reproduce the organelle of storage CP described in plants, but because of the large amount of ESCPR observed in all transfected cells, indicating the capacity for efficient accumulation of these cells. In addition, the different types of transfected cells showed the same localization and accumulation patterns as the fusion proteins (see Figures 2A, 2B and 2D), and this pattern appears independently of the target fused with the CPIS (Figures 2B and 2C). The cells were co-transfected with plasmid pDsRed2-RE (Clontech) containing the sequence for a fluorescent protein used as a marker of RE to analyze the subcellular origin of the induced CPLS. It is interesting, as can be seen in Figures 2D, 2E and 2F, that the RX3-Ct and the RE marker are colocalized in the ER and in assemblages similar to protein bodies, indicating the origin in ER of the ESCPR induced in cells of mammals as it occurs in plant cells.

Example 3: Accumulation of fusion proteins in transformed yeast cells Sequences encoding EGF and hGH were fused in the NX-terminal gamma zein RX3 coding sequence (WO2004003207) and introduced into vector pYX243 (R &D Systems) to obtain constructs d 17 and c1 18. These constructs encoding the fusion proteins RX-EGF and RX-hGH were introduced into Saccharomyces cerevisiae. Expression analyzes were performed by growth of transformants in a galactose-containing medium and equivalent amounts of cells and media were analyzed with SDS-PAGE and immunoblot using specific antibodies against the expressed recombinant proteins. As can be seen in Figure 3A both fusion proteins RX3-EGF (slots c1 17) and RX3-hGH (slots c1 18) accumulated in the yeast cells, and no traces of protein were detected in the media. The accumulation of hGH fusions and those derived from hGH was also studied in the yeast Pichia pastoris that was transformed with the c135 and c121 constructs (which encode the fusion protein RX3-hGH) and c136 (which encodes the hGH protein, see schematic representation in Figure 3B). The transformants that accumulate the highest levels of recombinant proteins were selected. Two different signal peptides were used to express the fusion protein, the zein gamma signal peptide (Fig. 3B, SPg) and the peptide factor alpha prepro (Fig. 3B, Afprepro). In addition, a secretion control was also analyzed using the peptide alpha factor prepro of the fused yeast directly in hGH (Fig. 3B, c136). The total proteins of the cells and media were analyzed with Western blot using antibodies specific against hGH cultured in rabbit. As expected, hGH was secreted into the medium (Fig. 3B, slot c136 / m) when the Afprepro peptide was used. In contrast, the RX3-hGH fusion protein accumulated within the yeast cells regardless of the signal peptide used. As can be seen in fig. 3B, the fusion protein was inside cells in both cases, when the yeast Afprepro peptide was used (slot c135 / y) and when the zein gamma signal peptide (slot c121 / y) was used, no traces were detected of the protein in the media (not shown). Thus, the proline-rich N-terminal domain of the gamma zein was sufficient to mediate the retention of protein in the endomembrane compartment of the yeast cells, and more specifically a dense fraction corresponding to CP-like structures that could be separated by centrifugation . The results obtained in Saccharomyces cerevisiae and Pichia pastoris are examples of other eukaryotic organisms apart from plants and the animal kingdom in which fusion proteins containing a seed storage protein are efficiently compiled and accumulated in CP-like structures.

Experimental procedures Plasmid constructs for transfection of mammals. The synthetic gene corresponding to the mature calcitonin sequence (Ct) was obtained as described (patent application WO2004003207). The synthetic gene encoding the 53 amino acids of active hEGF was obtained by the PCR method of overlapping the primer, using 4 oligonucleotides of about 60 bases, with 20 overlapping bases. The hEGF synthetic cDNA included a 5 'linker sequence corresponding to the specific cleavage site of Factor Xa. The oligonucleotides [EGF1 (SEQ ID NO: 25); EGF2 (SEQ ID NO: 26); EGF3 (SEQ ID NO: 27); and EGF4 (SEQ ID NO: 28) were purified with denaturing polyacrylamide gel. The cDNA sequence encoding the 191 amino acids of human growth hormone (hGH) was obtained from cDNA of the human pituitary gland (Clontech, BDBiosciences) by PCR using the oligonucleotides GH5 (SEQ ID N °: 29) and GH3 (SEQ ID N °: 30) which include the sequence corresponding to the enterokinase cutting site. Synthetic genes that correspond to mature calcitonin (Ct), WO2004003207) and hEGF sequences as well as the cDNA encoding hGH were fused to the coding sequence of N-terminal gamma zein RX3 (patent WO2004003207) and introduced into pUC18. The Sall-BamHI restriction fragments of the plasmids derived from pUC18, pUC18RXCt, pUC18RX01 and pUC18RX06, containing the corresponding fusion protein sequences of RX3-Ct, RX3-EGF and RX3-hGH were introduced into the vector pcDNA3.1- (Invitrogen) restricted with Xho l-Bam Hl. In the resulting constructs called p3.1 RX3CT, p3.1 RX3EGF and p3.1 RX3hGH, the fusion protein sequences were under the CMV promoter and the pA BGH terminator.

Plasmid constructs for transformation of yeast Vectors and host strains: Saccharomyces cerevisiae strain (genotype Mata his3 Ieu2 met15 ura3 bar1 :: URA3) was transformed by the use of constructs derived from vector pYX243 (promoter GAL, LEU2, AmpR, from R &D Systems). The strain of Pichia pastoris GS1 15. { his4) and the vectors pPIC9 and pPIC3.5K (promoter AOX1, HIS4, AmpR) were from Invitrogen life tech.

Plasmid constructs: Sall restriction fragments (blunt-ended) -BamHI from the plasmids described above derived from pUC18, pUC18RX01 and pUC18RX06, containing the corresponding fusion protein sequences RX3-EGF and RX3-hGH were introduced into vector pYX243 (R &D Systems) restricted with EcoRI (blunt ends) -Bam Hl. In the resulting constructs that are called, respectively, d 17 and d 18, the fusion protein sequences were under the control of the GAL inducible promoter. Sall restriction fragments (blunt ends) -BamHI (blunt ends) of the plasmids derived from pUC18, pUC18RX01 and pUC18RX06, were introduced into the vector pPIC3.5K (Invitrogen) restricted with Notl (blunt ends) -EcoRI (blunt ends) to obtain plasmids c120 and c121 to transform the Pichia Pastoris. Plasmid pPIC9 (Invitrogen) was used to analyze the expression of fusion proteins using a yeast signal peptide, the alpha prepro peptide of Saccharomyces. The sequences flanked with Xhol-Notl encoding the hGH and RX3-hGH proteins were obtained by PCR using pUC18RX06 as template and the oligonucleotides af06 (SEQ ID NO: 31), afRX (SEQ ID NO: 32) and 06Not (SEQ ID N °: 33). These sequences contained the sequence encoding the KEX2 site necessary for efficient cleavage of the alpha prepro peptide (Invitrogen, Pichia expression kit). PCR products were cloned in pPIC9 restricted with Xhol-Notl, resulting in plasmids c135 and c136 containing, respectively, the sequences of RX3-hGH and hGH proteins fused to the peptide factor alpha prepro.

Transformation of the yeast Saccharomyces cerevisiae strain (Ieu2) was transformed with the plasmids constructs c1 17 and c118 with the LiAc process (Ito et al 1983, J. Bacteriol 153: 163-168) and transformants were selected in Leu plates. . Expression analyzes were done by the growth of transformants in a galactose-containing medium. The strain of Pichia pastoris GS1 15. { his4) was transformed with the Pichia EasyComp Kit (from Invitrogen life tech.) with plasmids c120 and c121 Sacl linearized and coated in an RDB His- medium. The Mut phenotypes were determined by streaking the colonies on MD and MM agar plates. Expression tests were performed by growth of transformants in YPD medium for two days. Thereafter, the cells were pelleted and suspended in an MM medium for another 48 hours and methanol was added to a final concentration of 0.5% for 24 hours. The transformants that accumulate the highest levels of recombinant proteins were selected. The recipes of the media were made as described by Invitrogen (Pichia expression Kit).

Extraction of yeast proteins and Western blot S. cerevisiae and P. pastoris expressing recombinant fusion proteins were pelleted. The aliquots of the respective incubation media were precipitated and stored at -20 ° C for further analysis. The cell pellets were also frozen and after thawing, the cells were disrupted with normal procedures using glass beads and H medium (50 mM HCI-Tris pH 8.0, 150 mM NaCl, 5 mM EDTA, 200 mM DTT and inhibitors of protease). Equivalent amounts of cells and media were analyzed with SDS-PAGE and immunoblot using specific antibodies against the expressed recombinant proteins. Each of the patents and articles cited in this document are incorporated by reference. The use of the article "one" is intended to include one or more. The above description and the examples are intended to be illustrative and are not to be taken as limiting. Still other variations are possible within the spirit and scope of this invention and those skilled in the art will easily deduce them by themselves.

Claims (20)

1. A eukaryotic host cell of non-superior plants containing recombinant fusion protein within assemblies similar to recombinant protein bodies (ESCPR), said fusion protein containing two interlinked sequences in which a sequence is a heterologous protein body-inducing sequence with with respect to the host cell and the other is the sequence of a product of interest.

2. The host cell according to claim 1 wherein the density of the ESCPR is from about 1.1 to about 1.35 g / ml.

3. The host cell according to claim 1 wherein said fusion protein further includes a linker sequence between the protein body-inducing sequence and the sequence of the product of interest.

4. The host cell according to claim 1 wherein the inducing sequence of the proteinaceous body comprises a prolamine or a modified prolamin.

5. The host cell of claim 4 wherein the protein body inducing sequence is a prolamin sequence.

6. The host cell according to claim 5 wherein the prolamin sequence is zein gamma, alpha zein or the prolamin of rice.

7. A method for producing a fusion protein comprising the following steps: (a) transforming eukaryotic host cells that do not belong to higher plants with a nucleic acid sequence comprising (i) a first nucleic acid encoding a protein body-inducing sequence (CPIS) which is operably linked in the same reading frame to (ii) a second nucleic acid sequence comprising the nucleotide sequence encoding a product of interest polypeptide; and (b) maintaining the transformed host cells for a period of time and under culture conditions suitable for expression of the fusion protein and within assemblies similar to recombinant protein bodies (ESCPR).

8. The method according to claim 7, wherein the 3 'end of the first nucleic acid sequence (i) is linked to the 5' end of the second nucleic acid sequence (i).

9. The method according to claim 7 wherein said nucleic acid encodes the linker sequence between the CPIS and the polypeptide product of interest.

10. The method according to claim 7 wherein said host cells are fungal cells, in particular yeast cells.

11. The method according to claim 7 wherein said host cells are algae cells.

12. The method according to claim 7 wherein said host cells are cells of animals, in particular cells of mammalian animals.

13. The method according to claim 7 which includes the additional step of recovering the expressed fusion protein.

14. The method according to claim 7, wherein the protein body-inducing sequence comprises a prolamine or a modified prolamin.

15. The method according to claim 14 wherein the prolamin sequence is a modified prolamin comprising (a) a signal peptide sequence, (b) a sequence of one or more copies of the repeated hexapeptide domain PPPVHL (SEQ ID NO: 1) ) of the gamma zein protein, and (c) a sequence of all or part of the ProX domain of the gamma zein.

16. The method according to claim 14, wherein the prolamin sequence is zein gamma, alpha zein or the prolamin of rice.

17. The method according to claim 7, wherein the nucleic acid sequence used to transform the host cells is present in an expression vector that includes one or more regulatory sequences.

18. The method according to claim 17 wherein said one or more regulatory sequences include a promoter.

19. A recombinant nucleic acid molecule comprising a vector containing one or more regulatory sequences operably linked to an exogenous nucleic acid segment that defines a gene encoding a fusion protein comprising (i) a protein body-inducing sequence that is linked a (ii) a product of polypeptide interest.

20. The nucleic acid according to claim 19 wherein said one or more regulatory sequences of said vector includes a promoter suitable for directing the expression of the gene in a compatible eukaryotic host cell.