MXPA00002336A - Expression of gonadotropins in dictyostelium - Google Patents

Abstract

The present invention relates to gonadotropins expressed in i(Dictyostelium). The gonadotropins are found to be secreted in a biological active form. Expression of gonadotropins in i(Dictyostelium) provides an easy way to select for gonadotropin mutants.

Description

EXPRESSION OF GONADOTROPINES IN DICTYOSTELIUM The invention relates to gonadotropins or mutants thereof expressed in Dictyostelium, pharmaceutical compositions cining the same, a method for the preparation of gonadotropins as well as a method for the selection of gonadotropin mutants with superagonistic or antagonistic properties. Gonadotropins form a family of structurally related glycoprotein hormones. Normal members include chorionic gonadotropin (GC), follicle stimulating hormone (HEF), luteinizing hormone (LH) and thyroid stimulating hormone (TSH). HET, HL and H EF are present in most vertebrate species and are synthesized and secreted by the pituitary. In addition, GC has only been found in primates, including humans, and in horses and is synthesized by the placental tissue. Hormones are heterodimeric proteins of about 30 kD formed by a non-covalent association of a common subunit and a specific hormone of the β subunit. Within a species, the a subunit is essentially identical for each member of the gonadotropin family; they are also highly conserved from species to species. The ß subunits are different for each member, ie, GC, HEF, H ET and HL, but show considerable homology in the structure. Further, also ß subunits are highly conserved from species to species. In humans, the a subunit consists of 92 amino acid residues, while the β subunit varies in size for each member: 111 residues in HEFh, 121 residues in HLh, 118 residues in HETh, and 145 residues in GCh (Combarnous, Y. (1992), Endocrine Reviews, 13., 670-691, Lustbader, JW et al. (1993), Endocrine Reviews, 14, 291-311). The β-subunit of GCh is substantially greater than the other β-subunits in that it cins approximately 34 additional amino acids at the C-terminus referred to herein as the carboxy-terminal peptide (PCT). This PCT has four oligosaccharides linked to serine. Gonadotropins have important functions in a variety of bodily functions including metabolism, temperature regulation and reproductive processes. The HEF of hypophyseal gonadotropin, for example, plays a pivotal role in stimulating the development and maturation of follicles, while HL induces ovulation (Sharp, RM (1990), Clin Endocrinol., 33, 787-807; Dorrington and Armstrong (1979), Recent Prog. Horm. Res., 35., 301-342): Currently, HEF is applied clinically, either alone or in combination with HL, for ovarian stimulation, that is, the ovarian hyperstimulation for in vitro fertilization (IVF) and induction of ovulation in vivo in infertile anovulatory women (Insler, V. (1988), Int. J. Fertility, 33, 85-97, Navot and Rosenwaks (1988), J. Vitro Fert, Embryo Transfer, 5, 3-13), as well as for male hypogonadism. Human chorionic gonadotropin (hCG) is involved in the maintenance of pregnancy in the early stages after conception and also has important therapeutic applications. The two subunits of the heterodimers exhibit the majority of conserved intra-subunit sulfide bonds: five disulfide bridges in the a subunit and six disulfide bridges in the β subunit. The corresponding cysteine residues are completely conserved among all members of the gonadotropin family. The X-ray structure recently obtained from GCh shows that these disulfide bonds are involved in three-dimensional patterns called disulfide knots. Gonadotropins have three or four asparagine residues that can be N-glycosylated and have an important impact on their conformation and biological activity. In addition, the C-terminal peptide (PCT) of GCh can be O-glycosylated at four positions of serine. The main role of glycosylated PCT appears to be the prolongation of the circulating half-life of hCG. The biosynthesis of glycoprotein hormones is a highly complex process. In the last decade, it has been clarified that the bending, assembly and secretion of gonadotropins are assisted by a large group of chaperones and folding enzymes, which reside in the endoplasmic reticulum and the Golgi apparatus. Because both the a subunit and the β subunit contain a so-called cysteine node, it can be anticipated that the isomerase protein Disulfide plays a key role in facilitating the bending process. In addition, it has been shown that the N-linked oligosaccharide side chains are required for proper folding, disulfide formation and GCh secretion (Feng, W. et al. (1995) J. Biol. Chem. 270, 1851- 1859). The successive assembly of the two subunits in the dimer is an absolute prerequisite for the biological activity of the dimer. The elucidation of the functional determinants of the heterodimeric glycoprotein hormones is often hindered by unwanted side effects of mutations on the assembly of the subunits. To facilitate structure / function analysis studies, mutants have been produced in which the coding regions of the GCh β subunit and the common subunit are contacted via peptide or intersubunit disulfide bond separators. It has been shown that these subunits and β covalently linked to hCG are able to bend in biologically active conformations. Gonadotropins have been expressed in Chinese hamster ovaries (OHC) cells and their recombinant derivatives, have biological activities comparable to native hormones (Olijve, W. et al. (1996) Mol. Hum. Reprod. 2, 371 -382) . This indicates that these host cells contain all the accompanying and folding enzymes necessary to assemble the subunits and β of GCh and carry out all the post-translational modifications necessary to complete the biological activity. In addition, it has demonstrated that the use of OHC cells in combination with site-directed mutagenesis is a valuable tool for the elucidation of functional determinants in glycoprotein hormones (Puett, D. and others H. (1994) in Glycoprotein Hormones (Lustbader, J. W. Puett, D. &Ruddon, RW Eds.) Pp. 59-82, Springer-Verlag, New-York) and to design the new potential therapeutic analogues of these hormones (Fares, FA and others (1992), Proc Nati Sci USA 89, 4304-4308). Unfortunately, the use of mammalian cells such as OHC cells for the expression of gonadotropins of humans suffers from some limitations. Therefore, it is not possible to generate the high number of transformants necessary for studies involving random mutagenesis of protein domains. It has been demonstrated that the random mutagenesis of the selected domains in proteins is a valuable tool to identify the structural determinants for the binding and bioactivity of the receptors. In addition, the use of OHC cells is expensive and requires a lot of work. Therefore, there is a need for the expression of gonadotropins in a more robust expression host. The host cells derived from the lower organisms can meet the requirements mentioned above, but it is expected that the doubled complex of the gonadotropins will prevent the proper expression and secretion of the complex recombinant proteins.

Now, it has unexpectedly been found that Dictyostelium is capable of producing highly complex glycoprotein hormones. The soil amoeba Dictyostelium discoideum is an organism that provides an attractive alternative for the heterologous expression of human glycoprotein hormones. Although they can develop and transform as easily as the Saccharomyces yeast, they have some complex characteristics that resemble mammalian cells, such as glycosylation and chemotaxis. In addition, it has recently been shown that Dictyostelium provides a useful system for random mutagenesis approaches. However, differences have been found between the glycosylation of the proteins produced in Dictyostelium compared to the material produced by the OHC cells. It is known that glycosylation plays an important role in the function of hormones, and considering that most of the glycosylation is performed by Dictyostelium, galactose, N-acetylgalactosamine or sialic acids do not bind to the oligosaccharide side chains (Slade, M. et al. (1997) Biotech, Genet, Eng. Rev., 1_4, 1-35). Although post-translational modification is not identical to that in higher eukaryotes, it has also been found that gonadotropins produced in Dictyostelium are biologically active. It was found that proteins are able to bind to their receptor and to stimulate the production of cAM P in cells expressing the H L / GC receptor of humans. The heterogeneity of the expressed glycoproteins is greatly reduced, which leads to much more homogeneous preparations of isohormones. Therefore, the gonadotropins produced in Dictyostelium are chemically well defined in relation to the more complex produced gonadotropins of OH C. This is one of the main advantages of analytical maintenance and validation of consistency from one batch to another batch. Since glycosylation is a very important determinant for the in vivo half-life of gonadotropins, the production of gonadotropins in Dictyostelium provides a tool to produce non-mutated gonadotropins with well-defined in vivo half-lives. In addition, the combination of Dictyostelium expression with the treated protein facilitates the specialized formation of gonadotropins for various clinical applications. Due to the complex inter- and intra-molecular fold of the two subunits, the larger number of disulphide bridges, disulfide nodes and post-translational modifications, it is notorious that active gonadotropins can be expressed in Dictyostelium and that properly folded molecules can prepare according to the invention. The formation of the appropriate disulfide bond is a critical event in the folding and maturation of functional gonadotropins. Especially, the disulfide bond formation in the β subunit is critical: all disulfide bonds they are required for efficient combination and bending. The detailed studies for the folding of GCh revealed that the folding of the molecule does not proceed by a simple sequential route, but proceeds independently in the different domains of the molecule. Therefore, it was thought that cells from lower organisms were not able to properly secrete bent gonadotropins. The present invention provides the gonadotropins expressed in Dictyostelium. The use of Dictyostelium discoideum has the advantage that it is a well-studied organism. The vegetative amoeba is easy to develop and is not expensive, either in axenic culture or in a Gram negative bacterium. In addition, several transformation vectors have been described that are capable of directing the expression of foreign proteins. The gonadotropins according to the invention can be dimeric, that is, composed of two non-covalently bound subunits. Preferably, the gonadotropin is GCh or H EF. However, gonadotropins may comprise modifications generally known in the art. In a preferred modification of the gonadotropins according to the invention, the C-terminus of the amino acid sequence of one of the subunits is linked, optionally, through a binding portion, at the N-terminus of the amino acid sequence of the another subunit. Preferably, the portion of Binding is a complete or partial PCT unit or a variant thereof, or a repeated oligopeptide, e.g. , a Ser-Gly peptide repeated 5 times. Another modification of the gonadotropins according to the invention may be an extension of the α and / or β subunit and its respective N or C terminus with a complete or partial PCT unit or a variant thereof. The extension may comprise the respective PCT units in a single form or in multiple forms. Alternatively, a complete PCT unit or a partial PCT unit formed therefrom, may be inserted at the N or C terminus of said subunits. Again, another modification is the introduction of one or more non-native disulfide bridges. In addition, the gonadotropins according to the invention can be glycosylated or partially glycosylated. The partially glycosylated gonadotropins, according to the invention, can be obtained by site-directed mutagenesis whereby one or more glycosylation recognition sites in the gonadotropins are removed. Alternatively, the glycosylation pattern of the gonadotropins according to the invention, can be modified by the introduction of the additional glycosylation recognition sites and, optionally, the removal of one or more glycosylation recognition sites, resulting in a modified glycosylation of said gonadotropins. A glycosylation recognition site, as used herein, consists of the amino acid sequence Asn-X-Ser / Thr, where X can be any amino acid. As used herein, the GC subunits a and ß, H EF, HL and H ET, as well as the heterodimeric forms, have in general their conventional definitions and refer to the proteins having the amino acid sequences known per se in the material, or allelic variants thereof, in which refers to the pattern of glycosylation exhibited. The "native" forms of these proteins are those proteins that have the amino acid sequences isolated from the relevant vertebrate tissue and have these sequences known per se, or their allelic variants. These "variants" are those proteins that have deliberate alterations in the amino acid sequences in relation to the native proteins. The alterations may include deletions, insertions, substitutions and single or multiple combinations thereof, and may be produced by, for example, site-specific mutagenesis. As used herein, "PCT unit" refers to the amino acid sequence found at the carboxy terminus of the β-subunit of GCh extending from residue 1 12-1 18 to residue 14 of amino acids at termination C or a portion of it. A "complete" PCT unit contains 28-34 amino acids, which depend on the N-terminus of the PCT. A "partial" PCT unit is an amino acid sequence that is present between positions 112-118 to 145 inclusive, but having at least one amino acid deleted from the shortest complete PCT unit possible (amino acid 118-145). It is understood that the "multiple" PCT units encompass the random dispositions the complete PCT unit or the partial PCT unit or combinations of both. Methods for the construction of gonadotropin genes according to the invention are well known in the art (Sambrook et al., Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, latest edition). The techniques for site-directed mutagenesis, binding of additional sequences, PCR, and construction of suitable expression systems, for now, are all well known in the art. Portions or all of the DNA encoding the desired protein can be synthetically constructed using normal solid phase techniques, preferably to include restriction sites to facilitate binding. Suitable control elements for transcription and translation of the included coding sequence can be provided in the DNA coding sequences. The invention also provides a method for the expression of gonadotropins or mutants thereof in Dictyostelium. Said method according to the invention comprises the steps of: transforming a Dictyostelium strain with a recombinant plasmid vector comprising the DNA sequence encoding the gonadotropin genes or mutated genes under the control of Dictyostelium regulatory sequences cultivate the recombinant strain under conditions to allow expression of the DNA sequence and isolate the expressed protein. Preferably, the protein that is expressed is a single chain protein, i.e., the subunits are covalently connected through a separation molecule. More preferably, the gonadotropin is a single chain hCG. Another aspect of the invention is to provide a method for easily protecting mutated gonadotropins. Said method comprises: the random mutagenesis of gonadotropin genes the insertion of the mutated genes into the plasmid vector of Dictyostelium transformation of a strain of Dictyostelium with a recombinant plasmid vector the culture of the clones under conditions that allow the expression of the DNA sequence - the determination of the ratio of the binding receptor / transduction signal and - the isolation of the clones with a ratio deviation of the ratio determined by the wild-type gonadotropins.

Preferably, the mutated gonadotropins show a receptor binding that is equal to the binding of the native protein in its receptor. More preferably, the affinity of the mutated protein in its receiver is superior to its native counterpart. The signal transduction must be at least two times higher or lower, compared to the native protein. Preferably, the difference in amounts of signal transduction adds up to a factor of 10. Proteins that exhibit a higher ratio are useful as antagonists while proteins with a lower ratio can be used as super agonists. Methods for determining receptor binding as well as in vitro and in vivo assays to determine the biological activity of gonadotropins are well known. Random mutagenesis is not necessarily carried out on the complete gonadotropin gene, but instead can be carried out on a gene of a single subunit or a well-defined region such as, e.g., the determinant cycle. In order to introduce the random point mutations into gonadotropin genes, the amplification of the regions of interest can be used with DNA Taq polymerase. Because the Taq DNA polymerase lacks a 3'- 5 'exonucleolytic editing activity, this enzyme is a DNA polymerase prone to errors, with a measured error rate of 10"5 to an error of 10" 4 per synthesized nucleotide. Therefore, the use of PCR with the Taq DNA polymerase under essentially normal reaction conditions can be used to introduce the mutations (Zhou, Y. et al. (1991), Nucí Acids Res. 1_9, 52). Without However, the frequency of mutations using these conditions are adequate to mutagenize relatively large sequences, but not for small DNA fragments (<500 bp). The infidelity of Taq DNA polymerase can be increased by the addition of Mn2 + and the use of relatively high concentrations of dNTP and Mg2 + (Leungh, D. W. et al. (1989), Technique i, 1-15). An alternative method for adjusting the mutation frequency, which also offers the opportunity to influence the types of mutation, is the use of dITP in combination with the limiting quantities of one of the four NTPs (Spee, J. and others (1993), N ucí Acids Res. 21_. 777-778). For the mutagenesis of short white DNA (<50 bp), the use of the degenerate oligonucleotides resembles that of the method of choice (Kirchhoff, F. And Desrosiers, RC (1996), Meth. Molec. Biol. 57, 323- 333). Usually, the procedures have four steps. In step one, the region of interest is amplified by CPR (modified). In the second step, the amplified DNA is digested with a pair of restriction endonucleases that are cut at each end of the DNA sequence of interest. In the third step, the DNA fragment containing the DNA sequence of interest binds to the restriction endonuclease digested by the DNA vector. In the fourth step, the resulting recombinant DNA molecules are introduced into the cells by transformation or electroporation. The DNA vectors encoding any of the gonadotropins according to the invention are also within of the scope of the invention. The DNA vectors according to the invention can be obtained by operatively joining the DNA encoding the native gonadotropins or variants thereof into the DNA comprising the regulatory sequences of Dictyostelium. Optionally, these vectors could also contain regions having the origins of replication and / or sequences encoding the polypeptide facilitating extrachromosomal replication. Said vectors have the advantage that they can replicate extrachromosomally in the Dictyostelium host cell. As explained, the variant gonadotropins according to the invention can be agonists or antagonists, depending on the mutation site. The mutation site can lead to moderate changes in the conformation of the molecule. If the mutation site, v.gr. , selected in parts of the protein that is associated with the binding receptor and / or the signal transduction, the protein excreted according to the invention can lead to a partial or complete loss of the signal transduction activity. Said altered gonadotropins, wherein the properties of the binding receptor are retained, can be used as antagonists. Also, gonadotropins with enhanced signal transduction and binding activities can be selected. Agonist gonadotropins, according to the invention, can be used for the same clinical purposes as native gonadotropins. In addition, proteins can be used as diagnostic tools to detect the presence or absence of antibodies with with respect to native proteins in biological samples. They are also useful as control reagents in screening equipment to evaluate the levels of gonadotropin hormones in various samples. Antagonists can be used, v. gr. , in the treatment of tumors that depend on gonadotropin; H L / hCG antagonists prevent H L from arising during controlled hyperstimulation of ovaries and H EF antagonists for male contraception. Pharmaceutical compositions suitable in accordance with the invention comprise one or more gonadotropins according to the invention and a pharmaceutically suitable carrier. Pharmaceutically suitable carriers are well known to those skilled in the art and include, for example, sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextrin, agar, pectin, peanut oil, olive oil, sesame oil and water. In addition, the pharmaceutical composition according to the invention may comprise one or more stabilizers such as, for example, carbohydrates including sorbitol, mannitol, starch, sucrosedextrin and glucose, proteins such as albumin or casein, and pH-regulating solutions similar to phosphates. alkaline Suitable routes of administration are intramuscular injections, subcutaneous injections, intravenous injections or intraperitoneal injections, or oral and intranasal administration.

The following examples are illustrative and should not be considered as limiting the scope of the invention. LEGENDS OF THE FIGURES Figure 1: Plasmids and cloning strategy. (1A) Plasmid maps MB12n and MB12n / PsA. Both plasmids consist of four major fragments, which contain the maintenance sequences of E. coli (bluescript), blasticidin resistance gene under the control of the De Dd promoter (BSR), a cloning band with the De Dd promoter ( act15) and the terminator (2H3) and sequences for extrachromosomal maintenance in Dictyostelium (G4 / D5, G5 / D6). MB12n / PsA contains the sequence binding PsA (Table 1) inserted in the unique BglII site of MB12n. (1B) cloning diagram of JV158. The oligonucleotides used are shown by the horizontal arrows. The xxx shown in the oligonucleotide b20aarev indicate that the sequence substitutions are made to optimize the sequence for the Dictyostelium codon preferably. (1C) JV10PSA cloning diagram. All oligonucleotide sequences are given in Table 1. Figure 2: HLh binding activity, wild-type GCh and Single-chain GCh (see GCh) produced in Chinese Hamster Ovary (OHC) or Dictyostelium discoideum (Dd) cells for OHC cell membranes for stable expression of human HL / GC receptor. The membranes were incubated with GCh labeled with 125 I in the absence or presence of HLh variation concentrations, unlabelled wild-type GCh or single-chain hCG. The displacement curves are presented as the percentage of maximum binding in each dose of the unlabeled hormone. Figure 3: In vitro biological activity of HLh, hCG and wild-type hCG (see hCG) produced by Chinese hamster's Ovaries (OHC) or Dictyostelium discoideum (Dd) cells. Extracellular cAMPs were measured by specific RIA after stimulation of the OHC cells for stable expression of the human HL / GC receptor. Figure 4: In vitro biological activity of wild type hCG produced by Chinese Hamster Ovarian (OHC) or Dictyostelium discoideum (Dd) cells. Luciferase production was measured after 4 hours of incubation at 37 ° C and stimulation of the OHC cells for stable expression of the human HL / GC receptor and a reporter construct. Figure 5: In vitro biological activity of wild-type HEF produced in Chinese Hamster Ovary (OHC) or Dictyostelium discoideum (Dd) cells. Luciferase production was measured after 4 hours of incubation at 37 ° C and stimulation of the stable expression of OHC cells in the HEF receptor and a reporter construct. Figure 6: In vitro biological activity of wild-type hCG of the OHC cells and selected from the mutants of GCh produced by Dictyostelium discoideum (Dd). Luciferase production was measured after 4 hours of incubation at 37 ° C and stimulation of stable expression of OHC cells in the human HL / GC receptor and a reporter construct.

EXAMPLES Example 1 Design of Expression Constructs A gonadotropin mutant consists of a completely intact subunit connected via a decamer of peptide containing five replicates of Ser-Gly in the β chain that lacks only its C-terminating peptide, was selected for the expression in Dictyostelium. Two constructions were generated, which differ in their leader sequences. The first contains the natural leader sequence of the β subunit of GCh. To limit possible problems in mRNA translation due to the presence of a considerable amount (approximately 40% in the β subunit of hCG) of the codons used infrequently, the first 30 bases of the coding sequence are shaped in altered form to Dictyostelium using the preferred codon. For the construction of the second construct, it was considered that the proteins involved in the secretion pathway of mammalian cells, via the ER and Golgi, they may not be conserved among the different species. To facilitate transport in Dictyostelium, the leader peptide of the β subunit of humans was exchanged with a leader peptide of a Dictyostelium glycoprotein. Protein A of the pre-spores (PsA) is transported on the plasma membrane. This leader has previously been used to express secreted heterologous proteins (Dittrich, W. et al. (1994) Bio / Technology 12, 614-618). The expression plasmids are called JV158 (codons adapted with the leader peptide of the β subunit) and JV10PSA (with the leader peptide of PsA) and are derived from MB12n. MB12n is a Dictyostelium discoideum plasmid, maintained extra-chromosomally, of 8.25 kb, which contains a unique restriction site between a Dd promoter and a terminator. MB12n consists of 2.9 kb (a fragment of Clal-Hincll partial digestion) of p155d1 (Hughes, JE et al., (1994) Mol.Cell. Biol. 14, 6117-6124) which contains the Dd origin of replication and two Dd genes required for replication (G4 / D5 and G5 / D6), 2.95 kb of pBluescript (Strategene) for propagation in E. coli, 1.35 kb containing the blasticidin resistance gene between the Dina Actin promoter 15 and the Actin terminator 8 of Dd (from pBsr2, Sutoh, K. (1993), Plasmid 30, 150-154) and a 1.05 kb cloning tape containing the Dina Actin promoter 15, a single restriction site of BglII and the 2H3 terminator of Dd (from BS18.2H3, Kumagai A. and others (1989) Cell 57, 265-275). The BglII site in the blasticidin resistance gene has been removed by mutagenesis. The Figure 1a illustrates the relative position of these components in MB12n. Plasmid MB12n / PsA contains a binding sequence encoding the 19 amino acid PsA leader peptide (Early, EA et al. (1988) Mol.Cell. Biol. 8 3458-3466; Dittrich, et al. (1994) Bio / Technology 1_2, 614-618) cloned in the unique BglII site. The plasmid has a unique Ndel site in the 3 'part of the PsA sequence and a single 5' Bglll site of the 2H3 terminator, to facilitate "in frame" directional cloning. The adjacent BglII site in the Dd Actin promoter 15 was removed during cloning (see Table 1). Using the b20aarev and alphater primers (Table 1) a single-chain GCh was amplified by PCR from a plasmid containing the GCh 1 mutant [β- (1-111) - (Ser-Gly) 5-a- (1 -92), (Heikoop, JC, et al. (1997) Eur. J. Biochem. 245. 646-662). The resulting fragment was cloned into MB12n after digestion with BglII. The b20aarev primer was designed to optimize the first 10 amino acids of the β chain for the use of the codon in Dictyostelium. The same template was amplified with the bmature primers (Table 1) and alphater to produce a product that, after digestion with Ndel and BglII, was cloned into MB12n / PsA. The resulting plasmids were labeled JSV158 and JV10PSA, respectively (See Figure 1B and 1C), PCR was carried out with an Expanded High Fidelity system (Boehringer Manneheim), using the following cycle parameters: denaturation for 3 minutes at 94 ° C , then 25 cycles with 30 seconds at 94 ° C, 30 seconds at 37 ° C and 60 seconds seconds at 72 ° C. The PCR products were separated by gel electrophoresis, removed and purified with Qiaex II (Qiagen) before the restriction of digestion and cloning. All DNA sequences were analyzed and confirmed by dideoxy sequencing. Table 1: DNA sequences for the oligonucleotides and the binding of PsA. The sequence of PsA also shows the sequence of the leader PsA peptide, as well as the only restriction sites BglII and Ndel.

Oligo ID: Sequence: PsA: (BglII) NdeI BglII agatcagaa attccaacat acatttattg catattatc actattaaca tatgcaaatg cagatct M K F Q H T F I A L L S L L T Y A N A b20aarev: gcagatctat ggagatgttc caaggtctcc tccttttatt actcctcagc atgggtggta catgggcatc CAAG alphater: ccagatctaa tttgtc acatttaaga bmature: gtcatcgaca tatgcaaatg catccaagga gccgcttcg Example 2: Expression of single-chain GCh in Dictyostelium The Dictyostelium AX3 strain was developed at a density of 2x106 cells per ml in axenic medium before electroporation. The electroporation conditions were basically as described in (Mann SKO et al. (1994) in: Cell Biology: a Laboratory Handbook, JE Celis edt, Academic Press, Vol 1, pp. 412-452), using 1 μg of plasmid DNA for the electroporation of 107 cells. Plasmids JV158 and JV10PSA were transformed into Dictyostelium, each well was a control plasmid MB12n. After electroporation, the cells in a cuvette were seeded on a 10 cm plate. 12 hours after electroporation, blasticidin was added to a final concentration of 5 μg / ml. 24 hours after electroporation, the medium, which contains the dead cells, will be aspirated, the cells resuspended by pipetting into the medium with blasticidin, and the cells are distributed over the 24 wells in a 96-well microtiter plate. The 24 wells were diluted in series each of 10, 100, 1000 times. The medium containing blasticidin was replaced for 3 days. Positive wells were identified 5-9 days after they were planted, and the transformation efficiency was calculated from the dilution series. Normally, between 1 x 104 and 6 x 104 colonies per μg of DNA were obtained. The simple wells were then selected for further experiments. A single well containing 200 μl of medium, sufficient for a HLh analysis of DELFIA®, has a cross-reactivity of 100% with GCh. The analysis was based on the direct sandwich technique, in which the monoclonal antibody directed against a specific antigenic site on the β-subunit was immobilized. After binding of intact GCh (single chain) in the solid phase antibody, the europium-labeled antibodies were bound and quantified were directed against a specific antigenic site on the a subunit. The analysis was carried out as described by the manufacturer (Wallac Oy, Turku, Finland). The results clearly show that the single-chain, immunologically active HCG is produced from the expression constructs and not from the control plasmid. In addition, the amount of single chain GCh produced from JV158 (267 mU / ml) appears to be considerably higher than the amount produced from JV10PSA (4.9 mU / ml), suggesting that, in this case, the leader peptide of β-GCh it is more effective than the Dictyostelium leader peptide of PsA. The kinetics of single-chain GCh production were studied for transformed JV158 cells. Different densities of the transformed cells were seeded on the plates in an axenic medium, they were developed for confluence and they were maintained for several days, without any change of the medium. An aliquot of the medium was absorbed for 24 hours and analyzed for the presence of single chain GCh. The level of expression reached a maximum of 4 to 5 days after the cells reached confluence (data not shown). Because the cells they started to separate from the plate approximately 6-7 days after reaching the confluence, the medium was cultivated on day 5 after reaching confluence. Example 3: Single-chain hCG activity expressed in Dictyostelium The ability of single-chain GCh of Dictyostelium to bind to the human HL / GC receptor was determined by a competitive binding analysis with the hCG of the heterodimer. The OHC cells were expressed in the human HL / GC receptor (Jia, X. -C. and others (1991) Mol.Endocrinol., 5, 759-768) were incubated simultaneously with 125 I-GCh and the purified material. of the cell culture supernatant of Dictyostelium. The activity of the purified single-chain GCh binding receptor was quantified with a radioligand receptor shift assay on the membrane fractions isolated from exponentially developed cells. In a total volume of 0.5 ml of pH buffer (final composition of 10 mM Tris-HCl, 5 mM MgCl2, 0.1% bovine serum albumin, pH 7.4), a fixed amount of the membrane protein was incubated with 125 I-GCh (20,000 cpm, approximately 12 pM) and the increased amounts of the competing protein for 18 hours at room temperature. GCh labeled with 125l (NEX-106) was obtained from Du Pont de Nemours. The specific binding was 10-12% of the total radioactivity added. After the union of incubation and the free hormone was separated by centrifugation. The highly purified recombinant gonadotropins were used as normal. For the purification of hCG, Dictyostelium cell culture medium was recovered from large culture plates (22 x 22 cm). The purification was carried out with the aid of a programmable FPLC system (Pharmacia, Roosendaal, The Netherlands) using the UNICORN control and chromatography monitoring system (Pharmacia, Roosendaal, The Netherlands). Purification of single-chain GCh was achieved using a combination of hydrophobic interaction and immunochemotherapy with the monoclonal antibodies specific for the β subunit of HL / GC. For this purpose, 150 ml of medium with a single chain GCh content of 0.372 units / ml, determined by DELFIA®, was produced. Using this means, approximately 3 units of GCh from a single purified chain were obtained (-5% yield). All procedures were carried out at 4 ° C. Co-incubation with variable concentration of wildtype hCG, wild-type HLh or wild-type hCG exhibit the binding of 15l-hCG in a dose-dependent manner (Figure 2). The displacement curves indicate the material produced by Dictyostelium capable of binding to the human HL / GC receptor. The affinity for the receptor is comparable to the affinity of the single chain GCh produced by the OHC cells, which are shown to be considerably less effective in shifting the 125l-GCh binding than the GCh heterodimer.

Therefore, there seems to be no significant difference in the binding capacity between the single chain GCh produced by Dictyostelium compared to the OHC cells. The bioactivity of single-chain GCh of Dictyostelium was analyzed by examining its ability to stimulate cAMP production in OHC cells that express the human HL / CG receptor. The cells were incubated for 4 hours with increased hormone concentrations in the presence of 0.1 mM of 3-isobutyl-1-methylxanthine. The extracellular cAMP was determined by RIA (Immunotech). The results demonstrate that the single-chain hCG produced by Dictyostelium was more capable of activating the human HL / GC receptor resulting in the production of cAMP. Only a small difference in potency was observed between the single chain GCh produced in Dictyostelium compared to the material produced in the OHC cells (IC5o value approximately five times lower). Example 4: Expression and bioactivity of heterodimeric GCh in Dictyostelium We promote the expression of heterodimeric GCh in Dictyostelium For the expression of the α and β subunits of GCh, two plasmids were generated. Its overall structure and organization is essentially identical to that of the MB12n described in Example 1. In order to facilitate the expression of two independent plasmids in Dd, the cassette of blasticide resistance in MB12n was replaced with the 2.4 kb Kpnl-Xbal fragment of p155d1 (Hughes, J. E. et al. (1994) Mol.Cell. Biol., 14, 61 17-6124) which contains a cassette of neomycin, cloned in MB12neo. For the construction of the expression vector of subunit a, its natural cDNA sequence was produced by PCR using the primers that introduce a Bgl II restriction site both at the 5 'end and the 3' end of the fragment, so that they could be cloned in MB 12neo. For the construction of the expression vector for the β subunit of GCh, M B 12n was modified to contain another unique restriction site (Sph I) 3 'at the Bg l l l cloning site [see example 1]. The cDNA of the β subunit of GCh was amplified using a 5 'primer resulting in alteration of the first 30 bases of the coding sequence that make up the preferred Dictyostelium codon usage [see Example 1]. The primers also introduced appropriate restriction sites at the 5 '(Bgl ll) and 3' end (Sphl) of the fragment to facilitate directional cloning. The two expression plasmids were simultaneously transformed into Dictyostelium. After transformation, the cells were plated at the cyclone dilution. The clonal transformants were then selected and further developed for analysis. The amount of hCG secreted by Dictyostelium was determined as described by the manufacturer using a H Lh analysis of DELFIA® (Wallac Oy, Turku, Finland), which has a 100% cross-reactivity with GCh. The results clearly show that the immunologically active GCh is more produced by Dictyostelium. Although the presence of heterodimeric hCG in the medium was demonstrated by the detection of epitopes, additional experiments were needed to establish if the hCG produced by Dictyostelium is biologically active. The quantification was based on immuno-reactivity. The heterodimeric GCh bioactivity of Dictyostelium was analyzed by examining its ability to activate the human HL / GC receptor in a luciferase reporter analysis. The result demonstrates that the heterodimeric hCG produced by Dictyostelium is more capable of activating the human HL / GC receptor (Figure 4). In addition, its biocapacity (IC5o value approximately twice as high) can be compared to the bioactivity of wild-type GCh produced by the OHC cells. Example 5: Expression and bioactivity of heterodimeric HEF in Dictyostelium To investigate if Dictyostelium is also capable of producing other complex glycoprotein hormones, the production of HEF in this organism was studied. In line with the strategy for GCh (example 4), two expression plasmids were generated. For the construction of expression vectors for the ß subunit of HEF, the cDNA was amplified using the 5 'primer giving as result the alteration of the first 27 bases of the coding sequence make up the preferred codon of the Dictyostelium used and the cloning is carried out as described for the β subunit of GCh. After the transformation of Dictyostelium, the cells were seeded on plates at the cyonal dilution. The clonal transformants were then selected and further developed for analysis. The amount of HEF secreted by Dictyostelium was determined by a sandwich immunoassay [SS-artikel]. The results show that Dictyostelium produces active immune FSH.

In addition, heterodimeric HEF produced by Dictyostelium was also able to activate the HEF receptor of humans (Figure ). Example 6: Random mutagenesis of a selected region of hCG in Dictyostelium Because it was shown that Dictyostelium is capable of producing biologically active gonadotropins, we encourage the development of a random mutagenesis approach. For this purpose, two amino acids were selected in the GCh determinant curve that have been shown to be involved in receptor binding and signal transduction. Specific base substitutions are introduced into the site-specific mutagenesis and the combination of the PCR fragments that overlap the sequences as described using the normal CPR conditions. The primers were designed to alter amino acids 94 and 95 of the β subunit of GCh. The first two nucleotides of both codons were completely altered randomly (A, C, T or G), while the third base was restricted in G or T to minimize the percentages of the entered codons of challenge. The PCR fragments were separated and sub-cloned in pCR® 2.1 (Invitrogen, Leek, The Netherlands). The primers were chosen such that the subcloned PCR fragments contain a BglII site at the 5 'end and a Sphl site at the end 37 After CPR and cloning, the combination of the pCR ® 2.1 constructs were transformed to E. coli. Subsequently, the DNA was isolated from a combination of the 200 transformants and after the restriction of digestion the mutated fragments of BglII / Sphl were subcloned into MB12n containing the site BglII and Sphl. After plating the E. coli transformants of the MB12 plasmids containing the random mutant fragments, 400 colonies were pooled and the DNA was prepared. The Dictyostelium was transformed simultaneously with the expression vector for subunit a by electroporation. The blasticidin selection (10 μg / ml) was introduced 5 hours after electroporation. The next day, the cells were cloned in 96-well plates using 4-fold dilutions and a selection of neomycin (10 μg / ml) was initiated. The means replacement every 3-4 days, maintaining selective conditions. Positive wells were identified 11-14 days after electroporation, and transformation efficiency was calculated from the dilution series. Typically, approximately 500 transformants were obtained by electroporation of 107 cells with 1 μg of GCh a and GChβ vectors. The simple wells were then selected for further experiments. A single well contains 200 μl of medium. Higher amounts of medium were recovered for the purification of the larger culture plates (22 x 22 cm). The supernatants of 85 Dictyostelium clones were analyzed for the presence of immuno- and bio-activity. As controls, several wild-type GChs that produce clones and the non-transformed Dictyostelium clones are present in the 96-well plates. The wild-type and mutant GCh concentrations were measured using the HLh analysis of DELFIA® (Wallac Oy, Turku, Finland), which has a 100% cross-reactivity with GCh. Subsequently, the in vitro biological activity was determined on the human HL / GC receptor. Half of the 85 mutants analyzed showed the B / l ratio varying from 0.35 to 1.05. Taking into account the variations of 2 simple analyzes, it was considered that the activities of these mutants can be compared with wild-type GCh. Approximately 40% of the mutants showed a decreased B / l ratio. This could be foreseen, because mutated amino acids 94 and 95 have been shown to be involved in the union of the receiver and the activation. The fact that 11 clones show no production of hCG is likely due to the interference of the altered amino acids with the appropriate folding of the mutated β-polypeptide and / or the association with the subunit a or the fact that not all clones contain the expression construction of the subunit a and ß. Twenty clones with the variation of the B / l ratios were selected for detailed analyzes. The selected clones were further developed in 6-well plates until confluency and the cell culture supernatants were recovered after an additional 4 days. Four of the twenty clones produced no detectable hCG. This agrees with the results for these clones the initial screening. Among the different clones, considerably variable amounts of hCG were produced (116-2118 mU / ml). Cell culture supernatants were analyzed for the presence of in vitro biological activity of hCG on a scale of concentrations. All mutants showing B / I ratios on the wild-type GCh scale in the initial screening, also exhibited a wild type in vitro biological activity that was analyzed in greater detail. The four supernatants showed no immunological activity, nor could they activate the HL / GC receptor. Therefore, it is concluded that these clones do not produce hCG. In addition, all mutants with decreased B / l ratios in the initial screening also show a clear increase in IC50. Your B / l ratios vary from 2 times to >100 times less than the wild type GCh produced by Dictyostelium. The dose / response curves for eight of the mutants are shown in Figure 6. To correlate the biological activities observed with the mutations present in the β subunit, the mutated expression vectors were sequenced. Total DNA was isolated from the selected Dictyostelium clones and transformed for E. coli. For each Dictyostelium clone, a number of transformants was analyzed by colony PCR for the presence of the subunit plasmid a or the plasmid of the β subunit. Subsequently, the DNA was isolated from several transformants containing the plasmid of the β subunit and the sequence analysis was carried out on the mutated region using an automatic sequencer (Pharmacia). Therefore, most Dictyostelium clones contain only one type of mutation in the GChß expression vector. These clones produce a single type of mutant. In addition, it is concluded that most of the analyzed Dictyostelium clones contain unrelated sequences on position 94 and 95 of the β subunit, suggesting that the mutagenesis has been more random. Example 7: Mass spectrophotometric analysis of GCh rec of Dictyostelium discoideum Recombinant GCh, produced by Dictyostelium, was isolated from the culture supernatant by interaction chromatography Subsequently hydrophobic and immunochromatography with MoAB 119A that reacts with a common epitope in HL and GCh. The antibody was immobilized on NHS-Sepharose. The eluted acid, GCh rec purified was dialyzed against 50% acetonitrile and lyophilized. The protein was dissolved in 0.1% TFA and the mass spectrometry analysis was carried out with MALDI-TOF using superDHB as matrix. As a reference, pure rectal HCh produced by the OHC cells and isolated with the same purification procedure was used. Both the a subunit and the ß subunit of the Dictyostelium material had a lower molecular weight than the corresponding subunits of the OHC cells. The average values of the peaks being 11578 and 17351 D for the Dictyostelium subunits and 14013 and 23284 D for the OHC polypeptides. This observation indicates that the minor and / or short carbohydrate chains were present on the hormone that produces Dictyostelium. In addition, the peak width of the corresponding subunits differ considerably, ie 11110-12452 D (1342 D) for Dictyostelium against 12959-15591 D (2632 D) for the OHC chain a and 17006-18104 D (1098 D) for Dictyostelium ß against 20674-25208 D (4534 D) for the OH OHC chain. This indicates that the glycosylation of the Dictyostelium product is much less complex.

LIST OF SEQUENCES (1. GENERAL INFORMATION: (i) APPLICANT: (A) NAME: AKZO NOBEL N.V. (B) STREET: Velperweg 76 (C) CITY Arnhem (D) COUNTRY. The Netherlands (E) POSTAL CODE (C.P.): 6824 BM (F) TELEPHONE: 0412666379 (G) TELEFAX: 0412650592 (¡i) TITLE OF THE INVENTION: Expression of gonadotropins in Dictyostelium (iii) NUMBER OF SEQUENCES. 5 (iv) COMPUTER READABLE FORM: (A) TYPE OF MEDIUM: Flexible disk (B) COMPUTER: IBM compatible PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Relay # 1.0 , Version # 1.30 (EPO) (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 67 base pairs (B) TYPE: nucleic acid (C) THREAD FORM. Simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) CHARACTERISTIC: (A) NAME / KEY: CDS (B) LOCATION. 6..62 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: AGATC ATG AA TTC CAA CAT ACA TTT ATT GCA TTA TTA TCA CTA TTA 47 Met Lys Phe GIn His Thr Phe Me Ala Leu Leu Ser Leu Leu 1 5 10 ACA TAT GCA AAT GCA GATCT 67 Thr Tyr Wing Asn Wing 15 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 2: Met Lys Phe Gln His Thr Phe lie Wing Leu Leu Ser Leu Leu Thr Tyr 1 5 10 15 Wing Asn Wing (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 74 base pairs (B) TYPE: nucleic acid (C) THREAD FORM. Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 3: GCAGATCTAT GGAGATGTTC CAAGGTCTCC TCCTTTTATT ACTCCTCAGC 50 ATGGGTGGTA CATGGGCATG CAAG 74 (2) INFORMATION FOR SEQ ID NO: 4: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) THREAD FORM. Simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 4: CCAGATCTAA ACATTTAAGA TTTGTG 26 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) THREAD FORM. Simple (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: GCTATCGACA TATGCAAATG CATCCAAGGA GCCGCTTCG 39 Met Lys Phe Gln His Thr Phe He Ala Leu Leu Ser Leu Leu 1 5 10

Claims (7)

1. A gonadotropin or a mutant thereof that can be obtained by a heterologous expression in a Dictyostelium host.

2. The mutant gonadotropin of claim 1, characterized in that the subunits are covalently linked.

3. The gonadotropin according to claim 1 or 2, for use as a therapeutic substance.

4. The gonadotropin according to claims 1-3, characterized in that the gonadotropin is GCh or HEF.

5. A pharmaceutical composition comprising the gonadotropin according to claims 1 or 2, and a pharmaceutically acceptable carrier.

6. A method for producing a gonadotropin or a mutant thereof comprising the steps of: transforming a Dictyostelium strain with a recombinant plasmid vector comprising the DNA sequence encoding the gonadotropin genes or mutated genes under sequence control regulators of Dictyostelium. cultivate the recombinant strain under conditions to allow expression of the DNA sequence and isolate the expressed protein.

7. A method for selecting a gonadotropin mutant with super-agonistic or antagonistic properties comprising the steps of: random mutagenesis of gonadotropin genes insertion of the mutated genes into the Dictyostelium plasmid vector transforming a Dictyostelium strain with a recombinant plasmid vector cultivate the clones under conditions to allow expression of the DNA sequence determining the ratio of the binding receptor / transduction signal and - isolate the clones with a relationship deviating from the ratio determined by the wild-type gonadotropins.