WO1996014409A1 - Production de peptides recombinants, analogues de peptides naturels hydrophobes - Google Patents

Production de peptides recombinants, analogues de peptides naturels hydrophobes Download PDF

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Publication number
WO1996014409A1
WO1996014409A1 PCT/FR1995/001464 FR9501464W WO9614409A1 WO 1996014409 A1 WO1996014409 A1 WO 1996014409A1 FR 9501464 W FR9501464 W FR 9501464W WO 9614409 A1 WO9614409 A1 WO 9614409A1
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Prior art keywords
peptide
sequence
recombinant
aaa
acc
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PCT/FR1995/001464
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English (en)
French (fr)
Inventor
Thien Nguyen Ngoc
Hans Binz
Mathias Uhlen
Stefan Stahl
Per Ake Nygren
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Pierre Fabre Medicament
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Application filed by Pierre Fabre Medicament filed Critical Pierre Fabre Medicament
Priority to AU41200/96A priority Critical patent/AU704594B2/en
Priority to NZ296562A priority patent/NZ296562A/xx
Priority to JP8515108A priority patent/JPH10508479A/ja
Priority to EP95939336A priority patent/EP0791059A1/fr
Publication of WO1996014409A1 publication Critical patent/WO1996014409A1/fr

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • 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
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to recombinant peptides, analogues of natural peptides, and having conserved the biological activity of these natural peptides. These peptides can be expressed by different types of cells and their production is improved compared to that of the natural peptide.
  • peptide any substance composed of a chain of amino acids, that is to say oligopeptide, polypeptide and protein.
  • Peptides have a wide variety of biological properties, and to avoid the problems of contamination, in particular, linked to purification techniques from biological products, we have been led to produce them by genetic engineering.
  • peptides are excreted through the cell membrane, in order to obtain them in the extracellular medium or, in the case of Gram-negative bacteria, in the periplasmic space.
  • a system can be introduced into a cell, in particular a bacterium, allowing expression of the peptide in a form fused with a membrane anchoring sequence, so as to obtain the fusion product covalently linked to the membrane surface.
  • RSV respiratory syncitial virus
  • the respiratory syncytial virus (RSV) is the most common cause of respiratory diseases in newborns: bronchopneumopathies (bronchiolitis). WHO estimates 5 ⁇ million cases of RSV each year, including 160,000 deaths worldwide. There are two subgroups of the virus (subgroups ⁇ and B).
  • RSV is classified in the family of Paramyxoviridae, genus pneumovirus comprising a non-segmented ⁇ RN genome, of negative polarity, coding for 10 specific proteins.
  • RSV structural proteins for a vaccine, such as the envelope proteins called protein F (fusion protein) or protein G, a 22 Kd glycoprotein, a protein of 9.5 Kd, or the major capsid protein
  • Application WO 89/02935 describes the protective properties of the entire F protein of RSV, optionally modified in monomeric or deacetylated form. A series of fragments of the F protein have been cloned in order to search for their neutralizing properties.
  • RSV infections of the upper airways treatment is essentially based on symptomatic medications identical to those of other viral infections.
  • RSV infections of the lower airways treatment in infants is based on maintaining proper hydration, aspirating secretions and administering oxygen if necessary.
  • a positive effect has been observed with ribavirin, a nucleotide active in vitro against RSV.
  • ribavirin a nucleotide active in vitro against RSV.
  • the applicants have built an original vector system, also called a shuttle vector, which is functional in Esche chia coli and Staphylococcus xylosus: the vector contains a secretory sequence signal S, and an XM region of membrane anchoring of protein A origin. Staphylococcus aureus, with a cloning site between S and XM allowing the insertion of one or more genes.
  • the hydrophobicity of the molecule must in certain cases be modified. However, these modifications must not alter the biological, in particular immunogenic, properties of the product.
  • the subject of the present invention is a method for producing a recombinant biologically active peptide, analogous to a natural peptide having at least one hydrophobic region, characterized in that it comprises a step in which a cell a DNA construct, coding for a peptide whose amino acid sequence differs from the sequence of the natural peptide by at least one modification in said hydrophobic region, and comprising elements ensuring expression and secretion of said peptide by cell, and in that, after culturing the cells, the peptide and / or the cells carrying said recombinant peptide are recovered.
  • the amino acid sequence is modified at least in a region different from the transmembrane region of the natural peptide.
  • the modification should preferably take place in a region which is not essential for the biological activity of interest of the peptide, which must be maintained.
  • This process involves a recombinant DNA construct in which a functional secretory signal sequence is linked to a structural gene which has been altered in order to modify the structure which allows the recombinant product to cross the membrane of the host cell, then that the recombinant product of the original structural gene is not when linked to the same secretory signal sequence; and the structural modifications of the recombinant product should be carried out by genetic engineering by altering the localization of the recombinant product expressed in a host cell.
  • the modifications aim to modify the hydrophobicity of the recombinant product.
  • the subject of the invention is therefore a method for producing a recombinant peptide in which the structural modifications of the gene lead to a peptide in which at least one hydrophobic amino acid of the sequence of the natural peptide is replaced by a non-hydrophobic amino acid.
  • at least one hydrophobic amino acid is deleted from the sequence of the natural peptide.
  • the hydrophobic amino acid is chosen from the following group: Tryptophan, Phenylalanine, Proline, Valine, Alanine, Isoleucine, Leucine and Methionine.
  • Structural modifications of the gene can be made by insertion of nucleotides, or by deletion of nucleotides.
  • the constructions in which the structural modifications of the gene are made by substitution of nucleotides by site-directed mutagenesis are also included in the invention.
  • the structural modifications of the gene could change the localization in such a way that the recombinant product is exposed to the membrane surface of the cell by a covalent bond to the membrane anchoring part.
  • the structural modifications of the gene can change the location so that the recombinant product is secreted into the culture medium.
  • the invention therefore also relates to the construction of DNA which comprises a secretion signal sequence operably linked to the DNA sequence coding for the recombinant peptide, and ensuring the translocation of said peptide and its extracellular secretion.
  • the invention relates to a method characterized in that the DNA construction comprises a signal sequence operably linked to the DNA sequence coding for said peptide and allowing the translocation of the peptide across the membrane of the host cell and its membrane anchoring.
  • Another object of the invention is recombinant peptides capable of being obtained by the process, characterized in that they differ from the natural peptide by at least one modification in the hydrophobic region of the natural peptide. They may appear anchored to the surface of the host cell.
  • peptides will in particular be chosen from analogs of a protein of the RSV structure or of a fragment of such a protein; more particularly, the recombinant peptide comprises a sequence analogous to protein G of RSV, in group A or B, in particular comprised between residues 130 and 230 of protein G of RSV, having at least 80% homology.
  • Protein G is an RSV envelope glycoprotein, with a molecular weight between 84 and 90 Kd, poor in methionine.
  • the sequence of protein G differs for subgroups A and B of RSV; the terms "protein G sequence" when used in the present application, should be understood as referring to both the sequence of subgroup A or of subgroup B, when this is not specified differently.
  • the Applicant has demonstrated that the sequence between amino acids 130 and 230 of the natural protein G is particularly suitable for inducing effective protection against infection by RSV, subgroups A and B, without inducing the pathologies observed. with vaccines based on the whole virus inactivated by formalin, or observed with whole F and G proteins.
  • the means allowing the expression of the polypeptide are known to those skilled in the art and are adapted according to the bacteria used.
  • the DNA sequence is introduced in the form of a plasmid, such as a shuttle plasmid.
  • the RSV proteins have to date been expressed in different expression systems such as vaccinia virus, baculoviruses or adenoviruses. However, potential problems are associated with the presence of residual virus particles.
  • the method according to the present invention uses a commensal bacterium from humans, apathogenic and edible.
  • the bacteria can belong to the genus Staphylococcus.
  • Staphylococcus xylosus which is a bacterium used in the food industry for many years, and can be administered, alive, orally.
  • Expression systems of heterologous epitopes on the surface of S. xylosus have been described in particular by N'guyen et al in Gene, 1993, 128: 89-94.
  • the heterologous polypeptide is expressed on the surface of the membrane of the bacterium, in a conformation essentially identical to that of the corresponding epitope of the natural protein G.
  • the presentation of the recombinant protein on the membrane surface of the bacteria depends on its chemical nature and its peptide sequence.
  • the amino acid cysteine at positions 173 and / or 186 has been replaced by an amino acid which does not form of disulfide bridge, in particular serine.
  • Such a mutation promotes the formation of the disulfide bridge between the cysteine residues remaining in positions 176 and 182, which is critical for the immunogenicity of the sequence; it prevents the formation of disordered disulfide bridges.
  • Peptides useful for the implementation of such a process are those comprising in particular one of the sequences ID No. 3 or ID No. 4.
  • the amino acids phenylalanine corresponding to positions 163, 165, 168 and / or 170 of the sequence of protein G are replaced by an acid polar amine, especially serine.
  • This modification can be associated with the mutations mentioned above.
  • a polypeptide can in particular have the sequence ID No. 5.
  • the suppression of the hydrophobic region situated upstream of the critical loop formed by the disulfide bridge between the amino acids cysteine in positions 176 and 182 allows the recombinant protein to better cross the bacterial membrane and to correctly expose the immunodominant part on the membrane surface.
  • the sequence between the amino acids numbered 162 and 170 is deleted. More particularly, the sequence of the heterologous peptide expressed in the bacterium can include the sequence ID No. 6.
  • the invention also comprises a bacterium expressing a peptide or a protein, capable of being obtained by the method described in the present application. Said bacteria can be used alive or killed.
  • Polypeptides or bacteria having one or more of the above characteristics are useful as medicaments.
  • the invention comprises pharmaceutical compositions, characterized in that they comprise a polypeptide or a bacterium according to the invention in admixture with pharmaceutically acceptable adjuvants.
  • the live vector oral vaccine must include the modified protein which has the optimal conformation to induce the best protection against RSV. This is why the present invention also relates to an application of such a pharmaceutical composition to the preparation of an oral vaccine intended to prevent infections caused by the respiratory syncytial virus.
  • the subject of the invention is the nucleotide sequences coding for a recombinant peptide analogous to a natural peptide as described above; these sequences may also contain elements ensuring expression of the peptide in one or more specific host cells. These elements make it possible to target the cells in which the construction will be expressed during its administration to a mammal, human or animal.
  • These can be DNA or RNA constructs, which will preferably be incorporated into a vector.
  • Suitable vectors are in particular plasmids or viruses of the Adenovirus type, which can be formulated in pharmaceutical compositions with acceptable excipients.
  • the subject of the invention is nucleotide sequences coding for a polypeptide carried by a peptide sequence comprised between the amino acid residues 130 and 230 of the protein G of the respiratory virus or for a polypeptide having at least 80% of homology with said peptide sequence, and further comprising the means allowing the expression of the polypeptide on the surface of the membrane of a non-pathogenic bacterium of the genus Staphylococcus.
  • a DNA sequence coding for a recombinant peptide analogous to a natural peptide, the recombinant peptide sequence exhibiting at least one modification in the non-transmembrane hydrophobic region of the natural peptide, forms part of the invention.
  • the method according to the invention comprises the following steps: a. transforming the host cells with a recombinant DNA construct containing a signal sequence which is operably linked to a structural gene, and the latter is modified so that the recombinant product can be translocated across the membrane of the host cell ; b. fermenting said host cell to express the recombinant product; vs. recover the extracellular proteins secreted by the cells transformed by the constructs.
  • the invention also comprises a recombinant cell which contains a DNA sequence or a construct as defined above.
  • This host cell can be a bacterium, Gram + or Gram-, a yeast cell or a mammalian cell.
  • Particularly suitable bacteria are chosen from the group comprising Escherichia coli, Staphylococcus xylosus, Staphylococcus carnosus.
  • the DNA sequence can be integrated into the chromosome of the bacteria, Gram positive or Gram negative.
  • This recombinant DNA construct may contain the gene coding for protein G of amino acid 130 to 230 of human RSV subgroup A fused upstream and / or downstream of that of subgroup B.
  • a type of construction can be carried out with the gene coding for the amino acid protein 130 to 230 of bovine RSV belonging to subgroup A, or subgroup B.
  • FIG. 3 Schematic of shuttle vector constructions, above pSE'mlpl ⁇ BBXM, in (A) vector pSE'G2BBXM; (B) vector pSE'G2subBBXM; and (C) vector pSE * G2delBBXM;
  • FIG. 6 Diagram of the principle of construction of the secretion vectors from the respective shuttle vectors pSE'G2subBBXM and pSE'G2delBBXM whose descriptions are detailed in Example 4.
  • the products secreted from the culture medium of S are illustrated xylosus carrying vectors whose stop codon (T) has been inserted upstream of the XM membrane anchoring region;
  • Figure 7 Analysis by SDS-Page and immunoblot of the fusion proteins secreted by S. xylosus;
  • G2 Construction of G2 by assembly of synthetic genes: The gene coding for the amino acid region 130-230 of the glycoprotein G of the RS virus, named G2, where we have in addition changed two Cys residues at position 173 and 186 into Ser with respect to the original sequence, is obtained by techniques of assembly of genes in solid phase (Stahl S. et al., 1993. BioTechniques, .L4.424-434). The oligonucleotide sequence has been optimized by combining the usual codons of bacteria such as Ecoli and Staphylococcus.
  • the oligonucleotides were synthesized by phosphoramidite chemistry on the automatic DNA synthesizer (Gene Assembler Plus, Pharmacia Biotech) according to the manufacturer's recommendations.
  • the oligonucleotides to be fixed in the solid phase are biotinylated at the 5 ′ end by the reagent Biotin-on phosphoramidite (Clontech).
  • the other oligonucleotides are phosphorylated in 5 ′ by the phosphate-on amidite reagent (Clontech) according to the Clontech protocol.
  • the oligonucleotides are deprotected and are purified according to the recommendations of Pharmacia.
  • the biotinylated oligonucleotides are purified by reverse phase liquid chromatography (PEP RPC column, Pharmacia).
  • the gene is assembled in two parts: G2am (upstream) from AA 130 to 177 with two restriction sites BamHI and PstI in 5 'and 3' of the gene, G2av (downstream) from AA 177 to 230 with two sites of restriction PstI and Ba HI in 5 'and 3' of the gene.
  • the G2 gene is reconstituted by ligating the two fragments G2am and G2av via the PstI site.
  • the immobilized double strand comprises a BamHI restriction site and the strand complementary to that which is biotinylated has 6 to 15 nucleotides protruding from its 5 ′ side (phosphorylated) allowing the next oligonucleotide to come to hybridize.
  • Hybridization of the latter oligonucleotide is carried out by raising the temperature of the medium to 70 ° C. to avoid the formation of secondary structures. Ligation is done by adding T4 DNA ligase (Gibco BRL). Thus, the gene is constructed successively, taking the precaution at each cycle of rinsing the solid support in order to eliminate the excess of unbound oligonucleotides before adding the following oligonucleotide. The last ligated double strand contains a PstI restriction site.
  • the double strand is then released from its solid support by digesting it with the restriction enzymes BamHI and PstI and then ligated into the cloning and sequencing vector prit28 (Hultman et al., 1988, Nucleosides Nucleotides 7 629-638) digested by same enzymes: the resulting vector is pR! T28G2am of size 3067bp.
  • the nucleic acid sequence of G2am is determined by DNA sequencing on the ABI automatic sequencer, according to the manufacturer's recommendations (Applied Biosystem).
  • the double strand is released from the solid support by enzymatic digestion successively with PstI and HindIII, cloned in pRIT28: the resulting vector is named pRIT28G2av of size 3091 bp.
  • the nucleic acid sequence of G2av is determined by DNA sequencing on the ABI automatic sequencer, according to the manufacturer's recommendations (Applied Biosystem).
  • the two fragments G2am and G2av are ligated by the restriction site PstI and the gene formed is cloned into pRIT28 by the BamHI sites in 5 ′ and HindIII in 3 ′: the resulting vector is named pRIT28G2 of size 3220 bp.
  • the nucleic sequence of G2 is determined by DNA sequencing on the ABI automatic sequencer, according to the manufacturer's recommendations (Applied Biosystem).
  • the G2 fragment is digested with BamHI and HindIII and cloned into the shuttle vector: pSE'G2BBXM (7666 bp) (FIGURE 3). List of oligonucleotides required for gene assembly
  • HindlII TH12 (33mer): 5'-GCCGACCACC AAACCGGTCG ACTAAGCrTC ACA-3 ' HindlII TH13B (19mer): 5'-Biotin-CCCTGTGAAG CTTGGTTTG-3 'TH14 (32mer): 5'-CATAAACCGC AGACCACCAA ACCGAAAGAA GT-3' TH15 (32mer): 5'-GTGGTCGGCA CTTCTTTCGG TTTGGTGGTC TG 3 ' : 5'-AAAACCGACC TTCAAAACCA CCAAAAAAGA T-3 "TH17 (3 lmer): 5'-CGGTTTATGA TCTTTTTTGG TGGTTTTGAA C-3 'TH18 (30mer): 5'-GGGCAAAAA ⁇ ACCACGACCA AACCGACCAA-3' TH19 (3 lmer): 5 ' GTCCrGTITrrTGGTCGGTTTGGTCGTGGTTT-3 'TH20 (34mer): 5
  • the gene fragment where the four phenylalanine residues at positions 163, 165, 168 and 170 are replaced by Serines is generated by gene amplification (PCR), from the G2 gene inserted in the vector pRIT28, using as pairs of primers RIT27 / TNG73 and RIT28 / TNG72 (see FIGURE 2. (1)).
  • the primers TNG72 and TNG73 are complementary to a region of 19 nucleotides containing three of the four phenylalanines: Rit27: 5 , -GCnCCGGCr CGTATGlTGTGTG-3 'Rit28: 5'-AAAGGGGGAT GTGCTGCAAG GCC-3'
  • the primer TNG73 introduces the first three mutations (TTC in TCC) on the upstream fragment amplified with RIT27.
  • the primer TNG72 introduces the last three mutations (TTC in TCC) on the downstream fragment amplified with RIT28.
  • Five temperature cycles (9 'c, 15 sec; 50 ' c, 1 min; 72 " c, 1 min) followed by twenty cycles (9 " c, 1 5 sec; 60 "c, 15 sec; 72 'c, 15 sec)
  • the two amplified fragments are mixed in a single tube diluted in PCR buffer without primers and the extension reaction is carried out in five temperature cycles (96 'c, 15 sec; 54 * c, 30 sec; 72 * c, 1 min).
  • the extension product is diluted 1/100 in PCR buffer containing the primers RIT27 and RIT28 and the gene amplification is carried out in 30 temperature cycles (96 * c, 15 sec; 54 'c, 15 sec; 72 ' c, 30 sec).
  • the fragment is then digested with the restriction enzymes BamHI / HindIII and cloned into the vector prit28 digested with the same enzymes: pRIT28G2sub.
  • the nucleic sequence of G2Sub is determined by DNA sequencing on the ABI automatic sequencer device, according to the manufacturer's recommendations (Applied Biosystem).
  • the G2sub fragment is digested with BamHI and HindIII and cloned into the shuttle vector: pSE'G2subBBXM (7666 bp) (FIGURE 3).
  • G2del (see FIGURE 2. (2)) In the same way, the G2 gene fragment deleted from the part containing the 4 phenylalanine residues from aa 162 to aa 170 is generated by PCR in two fragments: upstream with the primers RIT27 / TH48 on the one hand and downstream with RIT28 / TH 1 1 on the other hand.
  • the primers TH1 1 and TH48 are complementary to 13 nucleotides.
  • TH 1 1 5'-GTGCCGAGCA GCATCTGCAG CA-3 '
  • the fragment is then digested with the restriction enzymes I.amlll / Hindlll cl cloned in the vector prit28 digested with the same enzymes: pRIT28G2del.
  • the nucleic sequence of G2del is determined by DNA sequencing on the ABI automatic sequencer device, according to the manufacturer's recommendations (Applied Biosystem).
  • the G2dcl fragment is digested with BamHI and HindIII and cloned into the shuttle vector: pSE'G2delBBXM (7639 bp) (FIGURE 3).
  • oligonucleotide linker (5'-AGCTTGGCTG TTCCGCCATG GCTCGAG-3 ', with the complementary strand) is inserted into the HindIII site of the plasmid pSZZmpl ⁇ XM (Hansson et al, 1992, J. Bacteriol 174: 4239-4245), thus creating two additional restriction sites Ncol and Xhol downstream of the HindIII site of the resulting vector pSZZmpl 8 (XhoI) XM.
  • BB gene fragment coding for 198 amino acids, named from the binding region of the serum albumin of the streptococcal protein G (Nygren et al, 1988. J. Mol.
  • TSB 12.5 g of Yeast extract
  • Chloramphenicol (20 ⁇ g / ml)
  • preheating overnight
  • S. xylosus transformed by the shuttle vector pSE'G2BBXM or pSE'G2subBBXM or pSE'G2delBBXM Gôtz et al. protocol, 1 81, J Bacteriol., 145: 74-81.
  • the proteins are purified by affinity: the supernatant is passed through an HSA-Sepharose affinity column (Human Albumin Serum). After rinsing the column, the proteins are eluted with a pH 2.7 acid buffer and lyophilized.
  • the proteins are separated on two identical SDS-PAGE gels (12%) with the precolored standard molecular size markers (Gibco BRL). A gel is colored with Coomassie Blue. The second is transferred to the ProblotTM membrane (Applied Biosystem) for the immunoblot with the specific anti-G l antibody (obtained from rabbit serum immunized with the G l peptide (aal 74-1 87) according to current protocols. 'immunization). See Figure 5.
  • the cultures of the recombinant S. xylosus bacteria are made as described above.
  • the bacteria are resuspended in a PBS solution at 0.1% Sodium Azide (w / v) at the final concentration estimated by optical density (600 nm) equal to the unit.
  • 30 ⁇ l of stock solution are aliquoted in each conical well of a microtiter plate, centrifuged at 550 g for 10 minutes at 4 ° C.
  • the bacterial pellet is resuspended in a volume of 150 ⁇ l of PBS solution containing rabbit serum polyclonal anti-G2 (titre 1/1 280 000) diluted at l / 200th, incubation for 30 minutes.
  • the bacterial cells are rinsed twice with PBS and incubated in 150 ⁇ l of a PBS solution containing anti-rabbit FITC (Sigma) diluted to 1/100 for a period of 30 minutes. After rinsing the cells twice with PBS buffer, it is resuspended in a Falcon tube containing 1 ml of PBS-Paraformaldehyde buffer 1% (w / v). The prepared samples are analyzed on the FACScan TM device (Becton Dickinson). The fluorescence distribution of each cell suspension is analyzed by LYSIS II TM software and is represented by fluorescence histograms. See Figure 4.
  • Termination codons upstream of the region coding for the XM membrane anchoring, as shown in FIG. 6.
  • a unique Xhol restriction site between BB and XM was used to insert a double strand of oligonucleotides coding for three termination codons (Ter) in both directions of orientation, with the introduction of an Aat II restriction site:
  • pSE'G2subBBXM and pSE'G2delBBXM are thus digested with Xho I and are ligated with the double strand of oligonucleotides previously phosphorylated at 5 '.
  • the resulting vectors are pSE'G2subBB [Ter] XM (7693 bp) and pSE'G2delBB [Tcr] XM (7666 bp) respectively.
  • FIG. 7 shows: A) the SDS-PAGE gel, the separation of the secreted proteins from S. xylosus, under reduced conditions, in 1 and in 2 respectively represent the proteins G2subBB and G2delBB at the expected size: 35.23 Kda and 34.28 Kda; in B) the immunoblot of the proteins shows that the antibody specific to the G region (aal 74-187) or G l of the RSV clearly recognizes the two secreted proteins. Very few proteolytic degradations have been observed.
  • FIG. 8 shows that the spectra of S. xylosus carrying shuttle vectors pSE'mpl ⁇ BBXM, pSE'G2subBBXM and pSE'G2delBBXM are displaced in the axis of the fluorescence intensity towards the right, that is to say towards the presence of heterologous antigens on the surface of the bacteria. While the spectra of S.
  • xylosus carrying shuttle vectors pSE'G2subBBTerXM and pSE'G2delBBTerXM are not displaced there, this indicates that the heterologous antigens are absent on the surface of the bacteria and that they have been found and purified from the culture medium.
  • Comassie blue staining SDS-Page gel of the fusion proteins extracted from the bacterial membrane and purified by affinity on the Albumin column of the different constructions:

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PCT/FR1995/001464 1994-11-07 1995-11-07 Production de peptides recombinants, analogues de peptides naturels hydrophobes WO1996014409A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
AU41200/96A AU704594B2 (en) 1994-11-07 1995-11-07 Production of recombinant peptides as natural hydrophobic peptide analogues
NZ296562A NZ296562A (en) 1994-11-07 1995-11-07 Recombinant peptides and secretory mehods for their preparation
JP8515108A JPH10508479A (ja) 1994-11-07 1995-11-07 天然疎水性ペプチドアナログとしての組換えペプチドの産生
EP95939336A EP0791059A1 (fr) 1994-11-07 1995-11-07 Production de peptides recombinants, analogues de peptides naturels hydrophobes

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FR9413307A FR2726576B1 (fr) 1994-11-07 1994-11-07 Production de peptides analogues de peptides hydrophobes, peptide recombinant, sequence d'adn correspondante
FR94/13307 1994-11-07

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU673650B2 (en) * 1995-01-10 1996-11-14 University Of Edinburgh, The Novel vectors and use thereof for capturing target genes
WO1999003987A2 (fr) * 1997-07-17 1999-01-28 Pierre Fabre Medicament Epitopes du vrs et anticorps les comportant, utiles dans le diagnostic et la therapie
EP0970115A1 (en) * 1996-06-05 2000-01-12 Biomolecular Research Institute Ltd. Viral peptides with structural homology to protein g of respiratory syncytial virus
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US7507413B2 (en) 2001-04-12 2009-03-24 Human Genome Sciences, Inc. Albumin fusion proteins
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AU673650B2 (en) * 1995-01-10 1996-11-14 University Of Edinburgh, The Novel vectors and use thereof for capturing target genes
EP0970115A1 (en) * 1996-06-05 2000-01-12 Biomolecular Research Institute Ltd. Viral peptides with structural homology to protein g of respiratory syncytial virus
EP0970115A4 (en) * 1996-06-05 2002-02-13 Biomolecular Res Inst Ltd VIRAL PEPTIDES WITH STRUCTURAL HOMOLOGY ON PROTEIN G FROM RESPIRATORY SYNCYTIAL VIRUS (RSV)
WO1999003987A2 (fr) * 1997-07-17 1999-01-28 Pierre Fabre Medicament Epitopes du vrs et anticorps les comportant, utiles dans le diagnostic et la therapie
WO1999003987A3 (fr) * 1997-07-17 1999-04-08 Pf Medicament Epitopes du vrs et anticorps les comportant, utiles dans le diagnostic et la therapie
FR2819810A1 (fr) * 2001-01-23 2002-07-26 Pf Medicament Peptides non glycosyles derives de la proteine g du vrs et leur utilisation dans un vaccin
WO2002058725A2 (fr) * 2001-01-23 2002-08-01 Pierre Fabre Medicament Peptides non glycosyles derives de la proteine g du vrs et leur utilisation dans un vaccin
WO2002058725A3 (fr) * 2001-01-23 2003-01-09 Pf Medicament Peptides non glycosyles derives de la proteine g du vrs et leur utilisation dans un vaccin
US8993517B2 (en) 2001-12-21 2015-03-31 Human Genome Sciences, Inc. Albumin fusion proteins
US9221896B2 (en) 2001-12-21 2015-12-29 Human Genome Sciences, Inc. Albumin fusion proteins
US9296809B2 (en) 2001-12-21 2016-03-29 Human Genome Sciences, Inc. Albumin fusion proteins

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AU704594B2 (en) 1999-04-29
CA2204511A1 (fr) 1996-05-17
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EP0791059A1 (fr) 1997-08-27
JPH10508479A (ja) 1998-08-25
FR2726576A1 (fr) 1996-05-10
FR2726576B1 (fr) 1997-01-31
AU4120096A (en) 1996-05-31
NZ296562A (en) 1999-11-29

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