EP1012290A1 - Expression von gonadotropinen in dictyostelium - Google Patents
Expression von gonadotropinen in dictyosteliumInfo
- Publication number
- EP1012290A1 EP1012290A1 EP98948936A EP98948936A EP1012290A1 EP 1012290 A1 EP1012290 A1 EP 1012290A1 EP 98948936 A EP98948936 A EP 98948936A EP 98948936 A EP98948936 A EP 98948936A EP 1012290 A1 EP1012290 A1 EP 1012290A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- dictyostelium
- hcg
- gonadotropin
- gonadotropins
- expression
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P5/00—Drugs for disorders of the endocrine system
- A61P5/06—Drugs for disorders of the endocrine system of the anterior pituitary hormones, e.g. TSH, ACTH, FSH, LH, PRL, GH
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P5/00—Drugs for disorders of the endocrine system
- A61P5/24—Drugs for disorders of the endocrine system of the sex hormones
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the invention relates to gonadotropins or mutants thereof expressed in Dictyostelium, pha ⁇ naceutical compositions containing the same, a method for the preparation of the gonadotropins as well as a method for the selection of gonadotropin mutants with superagonistic or antagonistic properties.
- the gonadotropins form a family of structurally related glycoprotein hormones. Typical members include chorionic gonadotropin (CG), follicle stimulating hormone (FSH), luteinizing hormone (LH) and thyroid stimulating hormone (TSH). FSH, LH and TSH are present in most vertebrate species and are synthesized and secreted by the pituitary. CG has so far been found only in primates, including humans, and in horses and is synthesized by placental tissue.
- CG chorionic gonadotropin
- FSH follicle stimulating hormone
- LH luteinizing hormone
- TSH thyroid stimulating hormone
- the hormones are heterodimeric proteins of around 30 kD formed by a non- covalent association of a common ⁇ -subunit and a hormone specific ⁇ -subunit.
- the ⁇ -subunit is essentially identical for each member of the gonadotropin family; it is also highly conserved from species to species.
- the ⁇ - subunits are different for each member, i.e. CG, FSH, TSH and LH, but show considerable homology in structure.
- the ⁇ subunits are highly conserved from species to species. In humans, the ⁇ subunit consists of 92 amino acid residues, whilst the ⁇ subunit varies in size for each member: 111 residues in hFSH,
- hCG 121 residues in hLH, 1 18 residues in hTSH and 145 residues in hCG (Combarnous, Y. (1992), Endocrine Reviews, 13, 670-691, Lustbader, J.W. et al. (1993), Endocrine Reviews, 14. 291-311).
- the ⁇ subunit of hCG is substantially larger than the other ⁇ subunits in that it contains approximately 34 additional amino acids at the C-terminus referred to herein as the carboxy terminal peptide (CTP). This CTP bears four serine linked oligosaccharides.
- the gonadotropins serve important functions in a variety of bodily functions including metabolism, temperature regulation and the reproductive process.
- the hypophyseal gonadotropin FSH for example plays a pivotal role in the stimulation of follicle development and maturation whereas LH induces ovulation (Sharp, R.M.
- FSH is applied clinically, either alone or in combination with LH, for ovarian stimulation i.e. ovarian hyperstimulation for in vitro fertilisation (IVF) and induction of in vivo ovulation 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.
- IVF in vitro fertilisation
- hCG human choriogonadotropin
- the two subunits of the heterodimer display many conserved intra-subunit disulfide bonds: five disulfide bridges in the ⁇ -subunit and six disulfide bridges in the ⁇ -subunit.
- the corresponding cysteine residues are fully conserved among all members of the gonadotropin family.
- the recently obtained X-ray structure of hCG shows that these disulfide bonds are involved in typical three-dimensional patterns called disulfide knots.
- the gonadotropins possess three or four asparagine residues that can be N- glycosylated and have an important impact on its conformation and biological activity.
- C-terminal peptide (CTP) of hCG can be O-glycosylated at four serine positions. The major role of the glycosylated CTP seems to be the prolongation of the circulatory half-life of hCG.
- the gonadotropins have been expressed in Chinese Hamster Ovary (CHO) cells, and their recombinant derivatives have biological activities comparable to the native hormones (Olijve, W. et al. (1996) Mol. Hum. Reprod. 2, 371-382). This indicates that these host cells contain all chaperones and folding enzymes necessary to assemble the ⁇ - and ⁇ -subunits of hCG and to perform all post-translational modifications necessary for full biological activity. Furthermore, the use of CHO cells in combination with site-directed mutagenesis has proven to be a valuable tool for elucidating functional determinants in the glycoprotein hormones (Puett, D. et al H.
- Dictyostelium is capable of producing the highly complex glycoprotein hormones.
- the soil amoebae Dictyostelium discoideum is an organism that provides an attractive alternative for heterologous expression of the human glycoprotein homiones. While it can be grown and transformed with the same ease as the yeast Saccharomyces, it has some of the complex features that resemble mammalian cells, such as glycosylation and chemotaxis. Furthermore, it has recently been shown that Dictyostelium provides a useful system for random mutagenesis approaches. Nevertheless, there have been found differences between the glycosylation of proteins produced in Dictyostelium compared to material produced in CHO cells.
- glycosylation plays an important role in hormone function, and whereas most glycosylation is performed by Dictyostelium, galactose, N-acetylgalactosamine or sialic acids are not attached to the oligosaccharide side chains (Slade, M. et al. (1997) Biotech. Genet. Eng. Rev., 14, 1-35). Though the post-translational modification is not identical to that in higher eukaryotes, gonadotropins produced in Dictyostelium also were found to be biologically active. The protein is found to be capable of binding to its receptor and to stimulate cAMP production in cells expressing the human LH/CG receptor.
- Dictyostelium In addition, combination of the expression in Dictyostelium with protein engineering facilitates tailor made gonadotropins for several clinical applications. Because of the complex inter and intra molecular folding of the two subunits, the large number of disulfide bridges, disulfide knots and post-translational modifications, it is remarkable that active gonadotropins can be expressed in Dictyostelium and that properly folded molecules can be prepared according to the invention. Proper disulfide bond formation is a critical event in the folding and maturation of functional gonadotropins. Especially the disulfide bond formation in the ⁇ subunit is critical: all disulfide bonds are required for efficient combination and folding.
- the present invention provides for gonadotropins expressed in Dictyostelium.
- Dictyostelium discoideum has the advantage that is a well-studied organism.
- the vegatative amoebae are easy and inexpensive to grow either in axenic culture or on Gram-negative bacteria.
- transformation vectors have been described which are capable of directing expression of foreign proteins.
- the gonadotropins according to the invention can be dimeric i.e. composed of two non-covalently bound subunits.
- the gonadotropin is hCG or FSH.
- the gonadotropins can comprise modifications generally known in the art.
- the C-terminus of the amino acid sequence of one of the subunits is linked, optionally through a linker moiety, to the N-terminus of the amino acid sequence of the other subunit.
- the linker moiety is a complete or partial CTP unit or variant thereof, or a repeated oligopeptide e.g. a 5 times repeated Ser-Gly peptide.
- Another modification of the gonadotropins according to the invention can be an extension of the ⁇ and/or ⁇ subunit at their respective N- or C-terminus with a complete or partial CTP unit or a variant thereof.
- the extension may comprise the respective CTP units in single or multiple forms.
- a complete CTP unit or partial CTP unit or multiple forms thereof can be inserted in the N- or C-terminus of said subunits.
- another modification is the introduction of one or more non- native disulfide bridges.
- the gonadotropins according to the invention may be either glycosylated or partially glycosylated.
- Partially glycosylated gonadotropins according to the invention can be obtained by site-directed mutagenesis whereby one or more of the glycosylation recognition sites in the gonadotropins are removed.
- the glycosylation pattern of the gonadotropins according to the invention can be modified by the introduction of 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, wherein X can be any amino acid.
- the ⁇ and ⁇ subunits of CG, FSH, LH and TSH as well as the heterodimeric forms have in general their conventional definitions and refer to the proteins having the amino acid sequences known in the art per se, or allelic variants thereof, regardless of the glycosylation pattern displayed. "Native" forms of these proteins are those proteins which have the amino acid sequences as isolated from the relevant vertebrate tissue, and have these known sequences per se, or their allelic variants thereof.
- variants are those proteins which have deliberate alterations in amino acid sequences relative to the native proteins.
- the alterations may include single or multiple deletions, insertions, substitutions and combinations thereof, and can be produced by, for example, site specific mutagenesis.
- CTP unit refers to the amino acid sequence found at the carboxy terminus of the ⁇ subunit of hCG which extends from amino acid 112-118 to residue 145 at the C-terminus or to a portion thereof.
- a "complete” CTP unit contains 28-34 amino acids, depending on the N-terminus of the CTP.
- a “partial” CTP unit is an amino acid sequence which occurs between positions 112-118 to 145 inclusive, but which has at least one amino acid deleted from the shortest possible complete CTP unit (amino acid 118-145).
- Multiple CTP units are understood to encompass tandem arrays of the complete CTP unit or partial CTP unit or combinations of both.
- the invention also provides for a method for the expression of gonadotropins or mutants thereof in Dictyostelium.
- Said method according to the invention comprises the steps of: transforming a strain of Dictyostelium with a recombinant plasmid vector comprising a DNA sequence encoding the gonadotropin genes or mutated genes under control of Dictyostelium regulatory sequences culturing the recombinant strain under conditions to allow expression of the DNA sequence and isolating the expressed protein.
- the protein to be expressed is a single-chain protein, i.e. the subunits are covalently connected through a spacer molecule. More preferably the gonadotropin is single-chain hCG.
- Another aspect of the invention is to provide a method to easily screen for mutated gonadotropins.
- Said method comprises : - random mutagenesis of gonadotropin genes insertion of the mutated genes in a Dictyostelium plasmid vector transforming a strain of Dictyostelium with said recombinant plasmid vector culturing clones under conditions to allow expression of the DNA sequence determining the receptor binding / signal transduction ratio and - isolating clones with a ratio deviating from the ratio determined for wild type gonadotropins.
- the mutated gonadotropins show a receptor binding which equals the binding of the native protein to its receptor. More preferably, the affinity of the mutated protein to its receptor is higher than its native counterpart.
- the signal transduction has to be at least two-fold higher or lower as compared to the native protein. Preferably, the difference in signal transduction amounts a factor 10.
- Proteins exhibiting a high ratio are useful as antagonists whereas proteins with a low ratio can be used as super agonists.
- Random mutagenesis need not to be performed on the complete gonadotropin gene but instead can also be carried out on a single subunit gene or a well-defined region such as e.g. the determinant loop.
- amplification of the region(s) of interest with Taq DNA polymerase can be employed.
- Taq DNA polymerase lacks a 3'-»5' exonucleolytic editing activity, this enzyme is an error-prone DNA polymerase, with a measured error rate of 10 "5 to 10 "4 error per nucleotide synthesized. Therefore, use of PCR with Taq DNA polymerase under essentially standard reaction conditions can be used to introduce mutations (Zhou, Y. et al. (1991), Nucl. Acids Res. 19, 52). However, the frequency of mutations using these conditions is adequate for mutagenizing relatively large sequences, but not for small DNA fragments ( ⁇ 500 bp).
- the infidelity of Taq DNA polymerase can be increased by addition of Mn 2+ and the use of relatively high concentrations of dNTP and Mg 2+ (Leungh, D.W. et al. (1989), Technique 1, 1-15).
- An alternative method for the adjustment of the mutation frequency is the use of dITP in combination with limiting amounts of one of the four dNTPs (Spee, J.H. et al. (1993), Nucl. Acids Res. 21, 777-778).
- the use of degenerate oligonucleotides seems to be the method of choice (Kirchhoff, F.
- step one the region of interest is amplified by (modified) PCR.
- step two the amplified DNA is digested with a pair of restriction endonucleases that cut at each end of the DNA sequence of interest.
- step three the DNA fragment containing the DNA sequence of interest is ligated with restriction endonuclease digested vector DNA.
- step four the resulting recombinant DNA molecules are introduced into the cells by transformation or electroporation.
- DNA vectors encoding any of the gonadotropins according to the invention are also within the scope of the invention.
- DNA vectors according to the invention can be obtained by operatively linking the DNA encoding the native gonadotropins or variants thereof to DNA comprising Dictyostelium regulatory sequences.
- these vectors also might contain regions which contain origins of replication and/or polypeptide-encoding sequences facilitating extrachromosomal replication.
- Such vectors have the advantage that they can replicate extrachromosomally in the Dictyostelium host cell.
- the variant gonadotropins according to the invention can be agonists or antagonists, depending on the mutation site.
- the mutation site may lead to subtle changes in the conformation of the molecule. If the mutation site e.g. is selected in parts of the protein that are associated with receptor binding and/or signal transduction, the excreted protein according to the invention may lead to a partial or complete loss of signal transduction activity.
- Such altered gonadotropins, wherein the receptor binding properties are retained can be used as antagonists. Also gonadotropins with improved binding and signal transduction activities may be selected.
- the agonist gonadotropins according to the invention can be used for the same clinical purposes as the native gonadotropins.
- the proteins can be used as diagnostic tools to detect the presence or absence of antibodies with respect to the native proteins in biological samples. They are also useful as control reagents in assay kits for assessing the levels of gonadotropin hormones in various samples.
- Antagonists can be used e.g. in the treatment of gonadotropin dependent tumors; LH/hCG antagonists to prevent LH surges during controlled ovarian hyperstimulation and FSH antagonists for male contraception.
- Suitable pharmaceutical compositions according to the invention comprise one or more of the gonadotropins according to the invention and a pharmaceutical acceptable carrier.
- Pharmaceutical acceptable carriers are well known to those skilled in the art and include, for example, sterile salin, lactose, sucrose, calcium phosphate, gelatin, dextrin, agar, pectin, peanut oil, olive oil, sesame oil and water.
- 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 buffers like alkaline phosphates.
- stabilizers such as, for example, carbohydrates including sorbitol, mannitol, starch, sucrosedextrin and glucose, proteins such as albumin or casein, and buffers like alkaline phosphates.
- Suitable administration routes are intramuscular injections, subcutaneous injections, intravenous injections or intraperitoneal injections, oral and intranasal administration.
- FIG. 1 Plasmids and cloning strategy.
- MB12n/PsA contains a PsA linker sequence (Table 1) inserted into the unique BgLII site of MB12n.
- IB Cloning diagram of JvT58.
- oligonucleotides used are shown by the horizontal arrows.
- the xxx shown in oligonucleotide b20aarev indicate that sequence substitutions have been made to optimize the sequence for Dictyostelium codon preference.
- FIG. 2 Binding activity of wildtype hLH, hCG and single chain hCG (sc hCG) produced in Chinese Hamster Ovary cells (CHO) or Dictyostelium discoideum (Dd) to membranes of CHO cells stably expressing the human LH/CG receptor.
- the membranes were incubated with 125 I-labeled hCG in the absence or presence of varying concentrations unlabeled wildtype hLH, hCG or the single chain hCG's. Displacement curves are presented as the percentage of maximal binding at each dose of unlabeled hormone.
- FIG. 3 In vitro biological activity of wildtype hLH, hCG and single chain hCG (sc hCG) produced in Chinese Hamster Ovary cells (CHO) or Dictyostelium discoideum (Dd). Extracellular cAMP was measured by specific RIA after stimulation of CHO cells stably expressing the human LH/CG receptor.
- CHO Chinese Hamster Ovary cells
- Dd Dictyostelium discoideum
- Figure 4 In vitro biological activity of wildtype hCG produced in Chinese Hamster Ovary cells (CHO) or Dictyostelium discoideum (Dd). Luciferase production was measured after 4 h incubation at 37 °C and stimulation of CHO cells stably expressing the human LH/CG receptor and a reporter construct.
- Figure 5 In vitro biological activity of wildtype FSH produced in Chinese Hamster Ovary cells (CHO) or Dictyostelium discoideum (Dd). Luciferase production was measured after 4 h incubation at 37 °C and stimulation of CHO cells stably expressing the human FSH receptor and a reporter construct.
- Figure 6 In vitro biological activity of wildtype hCG from CHO cells and selected hCG mutants produced by Dictyostelium discoideum (Dd). Luciferase production was measured after 4 h incubation at 37 °C and stimulation of CHO cells stably expressing the human LH/CG receptor and a reporter construct.
- Dd Dictyostelium discoideum
- a gonadotropin mutant consisting of a completely intact ⁇ -subunit connected via a peptide decamer containing five Ser-Gly repeats to the ⁇ -chain only lacking its C- terminal peptide, was selected for expression in Dictyostelium.
- the first one contains the natural leader sequence of the ⁇ -subunit of hCG. To limit possible problems in mRNA translation due to the presence of a considerable amount
- the proteins involved in the secretion route of mammalian cells via the ER and Golgi, may not be conserved between different species.
- the leader peptide of the human ⁇ -subunit was exchanged with a leader peptide of a
- Prespore protein A Dictyostelium glycoprotein, Prespore protein A (PsA), which is transported over the plasma membrane. This leader had been used previously to express secreted heterologous proteins (Dittrich ,W. et al. (1994) Bio/Technology 12, 614-618).
- the expression plasmids were called JV158 (adapted codons with ⁇ -subunit leader peptide) and JV10PSA (with the PsA leader peptide) and were derived from MB12n.
- MB12n is an 8.25 kb extra-chromosomally maintained Dictyostelium discoideum (Dd) plasmid, containing an unique restriction site between a Dd promoter and terminator.
- MB12n consists of 2.9 kb (a Clal -Hindi partial digest fragment) from pl55dl (Hughes, J.E. et al. (1994) Mol. Cell. Biol. 14, 6117-6124) containing the Dd origin of replication and two Dd genes required for replication (G4/D5 and G5/D6), 2.95 kb from pBluescript (Strategene) for propagation in E. coli, 1.35 kb containing the blasticidine resistance gene between the Dd Actine 15 promoter and the Dd Actin
- Dd Dictyostelium discoideum
- Plasmid MB12n/PsA contains a linker sequence encoding the 19 amino acid PsA leader peptide (Early, E.A. et al (1988) Mol. Cell. Biol. £ 3458-3466; Dittrich , et al (1994) Bio/Technology 12, 614-618).cloned in the unique BglLI site.
- the plasmid has an unique Ndel site in the 3' part of the PsA sequence and an unique Bglll site 5 1 of the 2H3 terminator, to facilitate 'in frame' directional cloning.
- the Bglll site adjacent to the Dd Actinl5 promoter was eliminated during cloning (see Table 1).
- Primer b20aarev Using primers b20aarev and alphater (Table 1) a single chain hCG was amplified by PCR from a plasmid containing hCG mutant 1 [ ⁇ -(l-l ll)-(Ser-Gly) 5 - ⁇ - (1-92), (Heikoop, J. C. et al. (1997) Eur. J. Biochem. 245, 656-662). The resulting fragment was cloned in MB12n after digestion with BglTI. Primer b20aarev is designed to optimize the first 10 amino acids of the ⁇ chain for codon usage in Dictyostelium.
- Fidelity (Boehringer Mannheim) system, 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 at 72 °C.
- PCR products were separated by gel electrophoresis, excised and purified with Qiaex II (Qiagen) before restriction digestion and cloning. All DNA sequences were analyzed and confirmed by dideoxy sequencing.
- Table 1 DNA sequences for oligonucleotides and PsA linker.
- the PsA sequence also shows the peptide sequence of the PsA leader, as well as the unique restriction sites Bglll and Ndel.
- Dictyostelium strain AX3 was grown to a density of 2x10 6 cells per ml in axenic medium before electroporation.
- the electroporation conditions were basically as described (Mann S.K.O. et al (1994) in: Cell Biology: a Laboratory Handbook, J.E. Celis edt, Academic Press, Vol 1, pp 412-452), using 1 ⁇ g plasmid DNA for electroporation of 10 7 cells.
- Plasmids JV158 and JV10PSA were transformed to Dictyostelium, as well as a MB12n control plasmid. After electroporation, the cells from one cuvette were seeded in a 10 cm plate. 12 hours after electroporation blasticidine was added to a final concentration of 5 ⁇ g/ml. 24 hours after electroporation the medium, containing dead cells, was aspirated, the cells were resuspended by pipetting in medium with blasticidine, and the cells were distributed over 24 wells in a 96 well microtiter plate. The 24 wells were each serially diluted 10, 100 and 1000 fold. The medium containing blasticidine was replaced every 3 days.
- a single well contains 200 ⁇ l of medium, sufficient for a DELFIA ® hLH assay which has a 100% cross-reactivity with hCG.
- the assay is based on the direct sandwich technique, in which monoclonal antibodies directed against a specific antigenic site on the ⁇ -subunit are immobilized. After binding of intact (single chain) hCG to the solid phase antibody, europium- labelled antibodies directed against a specific antigenic site on the ⁇ -subunit are bound and quantified. The assay is performed as described by the manufacturer (Wallac Oy, Turku, Finland).
- Dictyostelium leader peptide from PsA Dictyostelium leader peptide from PsA.
- the receptor-binding activity of purified single chain hCG was quantified with a radioligand receptor displacement assay on membrane fractions isolated from exponentially growing cells.
- a radioligand receptor displacement assay on membrane fractions isolated from exponentially growing cells.
- a fixed amount of membrane protein was incubated with 125 I-hCG (20,000 cpm, approximately 12 pM) and increasing amounts of competitor protein for 18 hours at ambient temperature.
- 125 I-labelled hCG (NEX-106) was obtained from Du Pont de Nemours. Specific binding was 10-12%. of the total radioactivity added. After incubation bound and free hormone were separated by centrifugation. Highly purified, recombinant gonadotropins were used as standards.
- hCG For purification of hCG from Dictyostelium cell culture medium was harvested from large (22 x 22cm) culture plates. Purification was performed with the aid of a programmable FPLC system (Pharmacia, Roosendaal, The Netherlands) using the control and chromatography supervision system UNICORN (Pharmacia, Roosendaal, The Netherlands). Purification of single chain hCG was accomplished using a combination of hydrophobic interaction and immuno chromatography with LH/CG ⁇ - subunit specific monoclonal antibodies. For this purpose, 150 mis of medium were produced with a single chain hCG content of 0.372 unit/ml as determined by
- the bioactivity of single chain hCG from Dictyostelium was analyzed by examination of its ability to stimulate cAMP production in CHO cells expressing the human LH/CG receptor. Cells were incubated for 4 hours with increasing concentrations of hormone in the presence of 0.1 mM 3-isobutyl-l-methylxanthin.
- the extracellular cAMP was determined by RIA (Immunotech).
- the expression vector for the ⁇ -subunit its natural cDNA sequence was produced by PCR using primers which introduce a BgUI restriction site both at the 5' and 3' end of the fragment, so that it could be cloned in MB12neo.
- MB12n was modified to contain another unique restriction site (SphI) 3' in the Bglll cloning site [see example 1].
- SphI unique restriction site
- the hCG ⁇ - subunit cDNA was amplified using a 5' primer resulting in the alteration of the first 30 bases of the coding sequence conform the Dictyostelium preferred codon usage [see example 1].
- the primers also introduced appropriate restriction sites at both the 5' (Bglll) and 3' (SphI) end of the fragment to facilitate directional cloning.
- the two expression plasmids were transformed simultaneously to Dictyostelium. After transformation, cells were plated to clonal dilution. Clonal transformants were then selected and further grown for analysis.
- the amount of hCG secreted by Dictyostelium was dete ⁇ nined as described by the manufacturer using a DELFIA ® hLH assay (Wallac Oy, Turku, Finland), which has a 100% cross-reactivity with hCG.
- DELFIA ® hLH assay Wilac Oy, Turku, Finland
- heterodimeric hCG was demonstrated in the medium by means of epitope detection, additional experiments were necessary to establish whether hCG produced by Dictyostelium is biologically active. Quantification was based on immuno-reactivity. The bioactivity of heterodimeric hCG from Dictyostelium was analysed by examination of its ability to activate the human LH/CG receptor in a luciferase reporter assay. The results demonstrate that the heterodimeric hCG produced by Dictyostelium is indeed able to activate the human LH/CG receptor ( Figure 4). Moreover, its bioactivity is comparable (IC 50 value approximately two-fold higher) to the bioactivity of wildtype hCG produced by CHO cells.
- Dictyostelium is capable of producing biologically active gonadotropins
- the pool of pCR ® 2.1 constructs was transformed to E.coli. Subsequently, DNA was isolated from a pool of 200 transformants and after restriction digestion the Bgi ⁇ /Sphl mutated fragments were subcloned in MB12n containing the Bglll and SphI site. After plating E.coli transformants of MB 12 plasmids containing the random mutant fragments, 400 colonies were pooled and DNA was prepared.
- Dictyostelium was transformed simultaneously with the expression vector for the ⁇ -subunit by electroporation. Selection with blasticidine (10 ⁇ g/ml) was introduced 5 hours after electroporation. The next day, cells were clonally diluted in 96 well plates using 4 fold dilutions and neomycin selection (10 ⁇ g/ml) was iniated. Medium was replaced every 3-4 days, maintaining selective conditions. Positive wells were identified
- the supernatants of 85 Dictyostelium clones were analysed for the presence of immuno- and bioactivity.
- As controls several wildtype hCG producing clones and non- transformed Dictyostelium clones were present on the 96-well plates.
- Concentrations of wildtype and mutant hCG were measured using a DELFIA ® hLH assay (Wallac Oy, Turku, Finland), which has a 100% cross-reactivity with hCG. Subsequently, the in vitro biological activity on the human LH/CG receptor was determined.
- Half of the 85 mutants analysed, show B/I ratios varying from 0.35 to 1.05.
- the mutated expression vectors were sequenced.
- the majority of Dictyostelium clones contains only one type of mutation in the hCG ⁇ expression vector.
- Recombinant hCG, produced by Dictyostelium was isolated from culture supernatant by subsequently hydrophobic interaction chromatography and immunochromatography with MoAb 119A that reacts with an epitope common to LH and hCG.
- the antibody was immobilised to NHS-Sepharose.
- the acid-eluted, purified rec hCG was dialysed against 50% acetonitrile and lyophilised.
- the protein was dissolved in 0.1%. TFA and mass spec analysis was carried out with MALDI-TOF using superDHB as matrix. Pure rec hCG produced by CHO cells and isolated with the same purification procedure was used as a reference.
- Both ⁇ - and ⁇ - subunits of the Dictyostelium material were of lower molecular weight than the corresponding subunits from CHO cells , the median values of the peaks being 11578 and 17351 D for the Dictyostelium subunits and 14013 and 23284 D for the CHO polypeptides. This observation indicates that less and/or shorter carbohydrate chains are present on the Dicyostelium-pvoduced hormone. Moreover, the peak width of the corresponding subunits differed considerably i.e.
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EP98948936A EP1012290A1 (de) | 1997-09-08 | 1998-09-02 | Expression von gonadotropinen in dictyostelium |
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EP97202757 | 1997-09-08 | ||
EP97202757 | 1997-09-08 | ||
EP98948936A EP1012290A1 (de) | 1997-09-08 | 1998-09-02 | Expression von gonadotropinen in dictyostelium |
PCT/EP1998/005717 WO1999013081A1 (en) | 1997-09-08 | 1998-09-02 | EXPRESSION OF GONADOTROPINS IN $i(DICTYOSTELIUM) |
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AU2001267337A1 (en) * | 2000-06-30 | 2002-01-14 | Maxygen Aps | Peptide extended glycosylated polypeptides |
EA009330B1 (ru) * | 2001-09-12 | 2007-12-28 | Апплайд Резеч Системз Арс Холдинг Н.В. | Способ содействия имплантации и/или снижения частоты выкидышей и набор для его осуществления |
YU21104A (sh) | 2001-09-12 | 2006-08-17 | Applied Research Systems Ars Holding N.V. | Upotreba hcg i lh u kontrolisanoj hiperstimulaciji ovarijuma |
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US5691455A (en) * | 1995-10-24 | 1997-11-25 | Akzo Nobel N.V. | Gonadotropins with non-native disulfide bridges |
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CN1269834A (zh) | 2000-10-11 |
AU746780B2 (en) | 2002-05-02 |
CA2302647A1 (en) | 1999-03-18 |
WO1999013081A1 (en) | 1999-03-18 |
NO20001178L (no) | 2000-05-05 |
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