AU704594B2 - Production of recombinant peptides as natural hydrophobic peptide analogues - Google Patents
Production of recombinant peptides as natural hydrophobic peptide analogues Download PDFInfo
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- AU704594B2 AU704594B2 AU41200/96A AU4120096A AU704594B2 AU 704594 B2 AU704594 B2 AU 704594B2 AU 41200/96 A AU41200/96 A AU 41200/96A AU 4120096 A AU4120096 A AU 4120096A AU 704594 B2 AU704594 B2 AU 704594B2
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- peptide
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Description
2 annually, resulting in 160,000 deaths globally. Two subgroups of the virus exist (subgroups A and B).
RSV is classified in the Paramyxoviridae family, genus pneumovirus, and has a non-segmented RNA genome of negative polarity which encodes 10 specific proteins.
Currently, no vaccine against RSV is available.
Inactivated virus vaccines have been shown to be ineffective and have sometimes even aggravated the infections of unweaned infants. Attempts in the 1960s to vaccinate with formalin-inactivated RSV led to failure: instead of conferring protection against reinfection with RSV, the vaccine had the effect of exacerbating the disease in infants.
Application WO 87/04185 proposed the use of RSV structural proteins for vaccine purposes, such as the envelope proteins termed protein F (fusion protein) or protein G, a glycoprotein of 22 Kd, a protein of 9.5 Kd or the major capsid protein (protein N).
Application WO 89/02935 describes the protection properties possessed by the entire RSV F protein, where appropriate modified in monomeric or deacetylated form.
A series of fragments of the F protein was cloned with a view to ascertaining their neutralizing properties.
However, the immunizing vaccines which have been tested to date have been found to be ineffective or have induced pulmonary pathology (bronchiolitis or peribronchitis) At the present moment, there is no basic treatment for infections due to RSV.
RSV infections of the upper airway: treatment is essentially based on symptomatic medication which is identical to that for other viral infections.
RSV infection of the lower airway: in unweaned infants, treatment is based on maintaining correct hydration, aspirating secretions and administering oxygen if required. A positive effect has been observed with ribavirin, a nucleotide which is active against RSV in vitro.
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In order to facilitate administration of the vaccine, it would be desirable to have available a product which is active orally, which engenders good immunity and whose side effects are reduced.
The applicants have constructed a novel vector system, also termed a shuttle vector, which functions in Escherichia coli and Staphylococcus xylosus: the vector encompasses a secretory signal sequence, S, and a membrane anchoring region, XM, which originates from Staphylococcus aureus protein A, and has a cloning site between S and XM into which one or more genes can be inserted.
In order to make it easier for the peptide to cross the membrane, the hydrophobicity of the molecule has to be modified in certain cases. Nevertheless, these modifications must not alter the biological, in particular immunogenic, properties of the product.
For this reason, the present invention relates to a process for producing a biologically active recombinant peptide which is an analogue of a natural peptide having at least one hydrophobic region, characterized in that it includes a step in which a DNA 15 construct, which encodes a peptide whose amino acid sequence differs from the sequence of the natural peptide by at least one modification in the said hydrophobic region and which includes elements which ensure that the said peptide is expressed and secreted by the cell, is introduced into a cell, and in that, after the cells have been cultured, the peptide and/or the cells harbouring the said recombinant peptide are recovered.
Accordingly, in one aspect the invention provides a process for secreting a biologically active recombinant peptide which is an analogue of a natural peptide having at least one hydrophobic region, characterized in that it comprises the steps of: a) introducing into a cell a DNA construct which includes: elements ensuring expression and secretion of the said peptide by the cell, and 25 a sequence encoding a peptide whose amino acid sequence differs from the sequence of the natural peptide by at least one modification in a nontransmembrane hydrophobic region of the peptide leading to the hydrophobicity reduction of the peptide; b) culturing the said cell; and c) recovering the said recombinant peptide and/or the cells harbouring the said recombinant peptide.
Preferably, the amino acid sequence is modified at least in one region which is different from the transmembrane region of the natural peptide.
2 MAW:PP:#25211.RS1
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26 February 1999 The modification should preferably take place in a region which is not essential for the biological activity of interest of the peptide, which should be preserved.
This process makes use of a recombinant DNA construct in which a functional secretory signal sequence o*
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MAW:PP:#25211.RS1 26 February 1999 4 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, whereas the recombinant product of the original structural gene is not allowed to do this when it is linked to the same secretory signal sequence; and the structural modifications of the recombinant product should be carried out by genetic manipulation while altering the location in a host cell of the expressed recombinant product.
According to one aspect of the invention, the modifications are directed towards modifying the hydrophobicity of the recombinant product.
The invention therefore relates to a process for producing 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. In another embodiment, at least one hydrophobic amino acid is, in the recombinant peptide, deleted from the sequence of the natural peptide.
Advantageously, the hydrophobic amino acid is selected from the following group: tryptophan, phenylalanine, proline, valine, alanine, isoleucine, leucine and methionine.
The structural modifications of the gene can be effected by inserting nucleotides or by deleting nucleotides.
Constructs in which the structural modifications of the gene are effected by substituting nucleotides by means of site-directed metagenesis are also included in the invention.
The structural modifications of the gene could be such as to change the location in such a way that the recombinant product is exposed at the membrane surface of the cell by means of a covalent linkage to the membrane anchoring part.
In another embodiment, the structural modifications of the gene can change the location such that the 5 recombinant product is secreted into the culture medium.
Theinvention also relates, therefore, to the DNA -construct which includes- a secretory signal sequence which is linked operationally to the DNA sequence which encodes the recombinant peptide and ensures translocation of the said peptide and its secretion outside the cell.
According to another aspect, the invention relates to a process which is characterized in that the DNA construct includes a signal sequence which is linked operationally to the DNA sequence which encodes the said peptide and which allows the peptide to be translocated across the membrane of the host cell and anchored to the membrane.
The invention also relates to recombinant peptides which can be obtained by the process and which are characterized in that they differ from the natural peptide by at least one modification in the hydrophobic region of the natural peptide. They can be anchored to the surface of the host cell. These peptides will be selected, in particular, from among the analogues of an RSV structural protein or of a fragment of such a protein; more especially, the recombinant peptide comprises a sequence which is an analogue of protein G of RSV, subgroup A or B, in particular between residues 130 and 230 of RSV protein G while exhibiting at least homology.
Protein G is an RSV envelope glycoprotein which has a molecular weight of between 84 and 90 Kd and is poor in methionine. The sequence of protein G differs in the A and B subgroups of RSV; when employed in the present application, the term "sequence of protein G" is to be understood to refer at one and the same time to the subgroup A sequence or the subgroup B sequence, when not otherwise specified.
The Applicant has demonstrated that the sequence encompassed between amino acids 130 and 230 of the natural G protein is particularly suitable for inducing effective protection against infection with RSV subgroups A and B without inducing the pathologies which are 6 observed with vaccines which are based on the whole formol-inactivated virus or observed with whole F and G -proteins.
The means for expressing the polypeptide are known to the person skilled in the art and are adapted in accordance with the bacterium employed.
Preferably, the DNA sequence is introduced in the form of a plasmid, such as a shuttle plasmid.
RSV proteins have previously been expressed in different expression systems such as vaccinia virus, baculovirus or adenovirus. However, potential problems are associated with the presence of residual viral particles.
In one of its embodiments, the process according to the present invention employs a bacterium which is a commensal of man, is non-pathogenic and is edible. In particular, the bacterium can belong to the genus Staphylococcus, i.e. Staphylococcus xylosus which is a bacterium which has been used in the food industry for many years and can be administered orally in the living state. Systems for expressing heterologous epitopes at the surface of S. xylosus have, in particular, been described by N'guyen et al. in Gene, 1993, 128: 89-94.
Preferably, the heterologous polypeptide is expressed at the surface of the membrane of the bacterium in a conformation which is essentially identical to that of the corresponding epitope of the natural protein G.
Presentation of the recombinant protein at the membrane surface of the bacterium depends on its chemical nature and on its peptide sequence.
The natural sequence of RSV protein G can be used and a DNA sequence can be introduced which encodes a peptide which comprises sequence ID No. 1 or sequence ID No. 2.
According to one aspect of the invention, in the sequence corresponding to the sequence encompassed between amino acids 130 and 230 of protein G, the amino acid cysteine in positions 173 and/or '186 has been replaced by an amino acid which does not form a 7 disulphide bridge, in particular serine.
Such a mutation promotes formation of the -disulphide bridge between the cysteine residues remaining in positions 176 and 182, which bridge is critical for the immunogenicity of the sequence; such a mutation avoids the formation of uncoordinated disulphide bridges.
Peptides which are useful for implementing such a process are, in particular, those which comprise one of the sequences ID No. 3 or ID N. 4.
According to another aspect of the invention, in the sequence of the heterologous polypeptide corresponding to RSV protein G, the phenylalanine amino acids corresponding to positions 163, 165, 168 and/or 170 of the protein G sequence are replaced by a polar amino acid, in particular serine.
This modification can be combined with the previously mentioned mutations. Such a polypeptide can, in particular, exhibit the sequence ID No. When implementing the process, suppression of the hydrophobic region which is situated upstream of the critical loop formed by the disulphide bridge between the cysteine amino acids in positions 176 and 182 enables the recombinant protein to cross the bacterial membrane more readily and to expose its immunodominant part correctly at the membrane surface.
According to yet another aspect of the invention, the sequence encompassed between amino acids numbered 162 and 170 is deleted in the peptide sequence corresponding to RSV protein G.
More especially, the sequence of the heterologous peptide expressed in the bacterium can comprise the sequence ID No. 6.
The invention also includes a bacterium which expresses a peptide or a protein which can be obtained by the process described in the present application. The said bacterium can be employed in the living or killed state.
Polypeptides or bacteria which exhibit one or more of the above characteristics can be used as 8 medicaments.
The invention includes pharmaceutical -compositions which are characterized in that they comprise a polypeptide or a bacterium according to the invention mixed with acceptable pharmaceutical adjuvants.
The oral vaccine which is based on a living vector should comprise the modified protein which exhibits the optimum conformation for inducing the best protection against RSV.
For this reason, the present invention also relates to application of such a pharmaceutical composition for preparing an oral vaccine which is intended to prevent infections which are caused by respiratory syncytial virus.
Finally, the invention relates to nucleotide sequences which encode a recombinant peptide which is an analogue of a natural peptide such as previously described; these sequences can additionally include elements which ensure expression of the peptide in one or more specific host cells. These elements enable the cells to be targeted in which the construct is to be expressed when it is administered to a human or animal mammal.
These sequences 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 it will be possible to formulate into pharmaceutical compositions together with acceptable excipients.
According to one of its aspects, the invention relates to nucleotide sequences which encode a polypeptide which is carried by a peptide sequence encompassed between amino acid residues 130 and 230 of respiratory syncytial virus protein G or which encode a polypeptide exhibiting at least 80% homology with the said peptide sequence, and which also include means for expressing the polypeptide at the surface of the membrane of a non-pathogenic bacterium of the genus Staphylococcus.
A DNA sequence which includes 9 a functional secretory signal sequence, a_ DNA sequence which encodes a recombinant peptide- which is an analogue of a natural peptide, with the recombinant peptide sequence exhibiting at least one modification in the non-transmembrane hydrophobic region of the natural peptide, is part of the invention.
The process according to the invention comprises the following steps: a. transforming the host cells with a recombinant DNA construct which encompasses a signal sequence which is operationally linked to a structural gene, with this latter being modified such that the recombinant product can be translocated across the membrane of the host cell; b. fermenting the said host cell in order to express the recombinant product; c. recovering the extracellular proteins which are secreted by the cells which have been transformed with the constructs.
The invention also includes a recombinant cell which harbours a DNA sequence or a construct such as previously defined.
This host cell can be a Gram-positive or Gramnegative bacterium, a yeast cell or a mammalian cell.
Particularly suitable bacteria are selected from the group comprising Escherichia coli, Staphylococcus xylosus and Staphylococcus carnosus.
The DNA sequence can be integrated into the chromosome of the gram-positive or gram-negative bacterium.
This recombinant DNA construct can encompass the gene which encodes protein G of human RSV subgroup A from amino acid 130 to 230 fused upstream and/or downstream of that of subgroup B.
One type of construct can be prepared using the gene which encodes the protein, from amino acid 130 to 230, of bovine RSV which either belongs to subgroup A or to subgroup B.
1U The following examples are intended to illustrate the invention without limiting its scope in any way.
In these examples, reference will be made to the following figures: Figure 1: Construction of plasmid pRIT28G2 down by gene assembly; -Figure 2: 1) Construction of gene G2 which is substituted by serine residues; 2) Construction of gene G2 from which residues 162 to 170 have been deleted; Figure 3: Construction of shuttle vector pSE'G2delBBXM; Figure 4: Analysis by flow cytometry of the recombinant surface proteins; Figure 5: Analysis by SDS-page of the proteins extracted from the membrane of recombinant S. xylosus; Figure 6 scheme of the construction principle of the secreted vectors from the corresponding shuttle vectors pSE'G2subBBXM et pSE'G2delBBXM the description of which are given in example 4. The products secreted from the growing culture medium of S. xylosus bearing vectors the stop codon of which having been inserted above the XM membrane anchoring region.
Figure 7: Analysis, by SDS page and immunoblot, of the fusion proteins secreted by S. xylosus; -Figure 8: Analysis by flow cytometry of the proteins secreted by S. xylosus harbouring different shuttle vectors.
EXAMPLES
EXAMPLE 1: I) Construction of G2 by assembling synthetic genes: The gene encoding the region encompassing amino acids 130-230 of glycoprotein G of RS virus, termed G2, in which, in comparison to the original sequence, we additionally changed two Cys residues at position 173 and 186 into Ser, is obtained by solid-phase gene assembly techniques (Stahl S. et col., 1993. BioTechniques, 14:424-434). The sequence of the oligonucleotides was optimized by combining the common codons of bacteria such as E. coli and Staphylococcus.
11 The oligonucleotides were synthesized by phosphoramidite chemistry on an automated DNA synthesizer -(Gene Assembler Plus, Pharmacia Biotech) according to the manufacturer's recommendations. The oligonucleotides to be bound to the solid phase are biotinylated at the end with the reagent Biotin-on phosphoramidite (Clontech). The other oligonucleotides are phosphorylated at the 5' end with the reagent Phosphate-on amidite (Clontech) according to the Clontech protocol. The oligonucleotides are deprotected and are purified in accordance with Pharmacia's recommendations. The biotinylated oligonucleotides are purified by reverse phase liquid chromatography (PEP RPC column:, Pharmacia).
The gene is assembled in two parts: G2up (upstream), from amino acid 130 to 177 with two restriction sites, BamHI and PstI, 5' and 3' of the gene, G2down (downstream), from amino acid 177 to 230 with two restriction sites, PstI and BamHI, 5' and 3' of the gene.
The G2 gene is reconstituted by ligating the two fragments G2up and G2down by means of the PstI site.
a) Assembling the G2up gene (FIGURE 1): In a micro tube, two complementary oligonucleotides, one of which is 5'-biotinylated: THIB CCGGATCCT ATGACCGTGA A-3' and TH2 TTCACGGTCA TAGGATCCGG-3', are hybridized and immobilized on magnetic beads which are coupled to streptavidin (Dynal, Oslo, Norway). The immobilized double strand includes a BamHI restriction site, and the strand which is complementary to the biotinylated strand possesses an overhang of from 6 to 15 nucleotides at its 5'end (phosphorylated) to which the following oligonucleotide can hybridize. Hybridization of this latter oligonucleotide is carried out while raising the temperature of the medium to 70 0 C in order to avoid formation of secondary structures. Ligation is accomplished by adding T4 DNA ligase (Gibco BRL). The gene is constructed successively in this manner while taking the precaution, in each cycle, of rinsing the solid support in order to eliminate 12 excess non-bound oligonucleotides before adding the following oligonucleotide. The last double strand to be -ligated on contains a PstI restriction site.
The double strand is then released from its solid support by digesting it with restriction enzymes BamHI and PstI and then ligated into the cloning and sequencing vector pRIT28 (Hultman et al., 1988, Nucleosides Nucleotides 7:629-638) which has been digested with the same enzymes: the resulting vector is pRIT28G2up, which is 3067bp in size. The nucleotide sequence of G2up is determined by DNA sequencing on an automated
ABI
sequencer in accordance with the manufacturer's recommendations (Applied Biosystems).
b) Assembling the G2down gene (FIGURE 1): In the same way, the G2down gene is solid-phase assembled on magnetic beads with the two first complementary oligonucleotides, one of which is TH13B (5'-biotin-TGTGAAGCTT AGTCGACCGG TTTG-3') and TH12 (5'-GCCGACCACC AAACCGGTCG ACTAAGCTTC ACA-3'), and so on. The double strand is released from the solid support by successive enzymic digestion with PstI and HindIII and cloned into pRIT28: the resulting vector is termed pRIT28G2down and is 3091bp in size. The nucleotide sequence of G2down is determined by DNA sequencing on an automated ABI sequencer in accordance with the manufacturer's (Applied Biosystems) recommendations.
c) Construction of the G2 gene G2up G2down: The two fragments, G2up and G2down, are ligated by means of the PstI restriction site, and the gene which has been formed is cloned into pRIT28 using the site and 3' HindIII site: the resulting vector is termed pRIT28G2 and is 3220bp in size. The nucleotide sequence of G2 is determined by DNA sequencing on an automated ABI sequencer in accordance with the manufacturer's (Applied Biosystems) recommendations. The G2 fragment is digested with BamHI and HindIII and cloned into shuttle vector:
M
13 pSE'G2BBXM (7666bp) (FIGURE 3).
List of oligonucleotides which are required for -assembling the G2 genes: BamHI THIB (2Omer) :5'-Biotin-CCGGATCCT ATGACCGTGA A-3' BarnHI TH2 (3Omer) S'-GTFFFFGGTFFFECACGGTCA TAGGATCCGG-3' TH3 (2 8mer) 5'-AACCAAAAAC ACCACGACCA CCCAGACC-3' TH4 (3 liner): 5'-G-1TfGCGG CTGGGTCTGG GTGGTCGTGG T-3' (3 liner) 5 t -CAGCCGAGCA AACCGACCAC CAAACAGCGTC-3' Th6 (29iner) 5 '-CGGThFG1TC TGACGCTUTTGGTGGTCG-3' TH7 (29mer): 5'-AGAACAAACC GCCGAACAAA CCGAACAAC-3' TH8 (34mer): 5'-JFFCGAAATG GXAATCGTTG 1TCGGTrTGT TCGG-3' TH9 (27mer) 5'-GATTCCATT TCGAAGTGT CAACTTC-3' Pst I TH 10 (33mer): 5'-TGCTGCAGAT GCTGCTCGGC ACGX-AG1TGA ACA-3' Pst I TI-Ill (2 2mer) 5'-GTGCCGAGCA GCATCTGCAG CA-3' HindIlI TH 12 (3 3mer) 5'-GCCGACCACC XAACCGGTCG ACTA-AGCTTC ACA-3' HinclI TH13 B (Il9mer) :5'-Biotin-CCCTGTGAAGCIGGTTTG-3' TH14 (32iner): 5'-CATAAACCGC AGACCACCAA ACCGAAAGAA GT-3' (3 2rer) 5 '-GTGGTCGGCA CTTCTICGG TTTGGTGGTC T-1r3' TH 16 (3l1iner): 5'-AAAACCGACC TITCAAAACCA CCAAAAAAGA T-3' TH 17 (3 liner) 5 '-CGG1TI'ATGA TC-FFFFITGG TGGWFFGAA G-3' TH 1 8 3mer) 5 '-GGGCAAAAA ACCACOACCA XACCGACCAA-3' Thi 9 (3 1 mer) S '-GTCGGTFFIT TGGTCGGTL GGTCGTGGTT T-3 (34mer): 5'-GGGCGATCAG CAAACCTATC CCGAACAAAA AACC-3
T
TIQ 1 (3 3iner) S'-T11GCCCGG TFT=G1TC GGGATACGTT TGC-3' Pst I TH22(2Siner) 5'-ATC TGCAGCAACA ACCCGACCTG CT-3' Pst I TH23 (34mer): 5'-TGATCGCCCA GCAGGTCGG;G TfTGTTGCTGC AGAT-3' I-11 Construction of G2sub by means of site-directed mutacrenesis: The gene fragment in which the four phenylalanine residues in'positions 163, 165, 168 and 170 are replaced with serines is generated, by gene amplification (PCR), from the G2 gene which is inserted into the vector pRIT28 14 using RIT27/TNG73 and RIT28/TNG72 as primer pairs (see FIGURE 2. The primers TNG72 and TNG73 are -complementary to a region of 19 nucleotides which encompasses three of the four phenylanalines: RIT27: 5'-GCTTCCGGCT CGTATGTTGT GTG-3' RIT28: 5'-AAAGGGGGAT GTGCTGCAAG GCG-3' TNG72 5'-C CAT TCC GAA GTG TCC AAC TCC GTG CCG AGC AG-3' TNG73 3'-GC TTG CTA AGG GTA AGG CTT CAC AGG On the one hand, primer TNG73 introduces the first three mutations (TTC to TCC) in the upstream fragment which is amplified together with RIT27. On the other hand, primer TNG72 introduces the last three mutations (TTC to TCC) into the downstream fragment which is amplified together with RIT28. Five cycles of temperatures (96°C, 15 sec; 50 0 C, 1 min; 72°C, 1 min) are followed by five cycles of (96°C, 15 sec; 60°C, 15 sec; 72°C, 15 sec). The two amplified fragments are mixed in a single tube and diluted in PCR buffer without primers, and the extension reaction is carried out in five cycles using the following temperatures (96°C, 15 sec; 54°C, sec; 72 0 C, 1 min). The extension product is diluted 1/100 in PCR buffer containing the RIT27 and RIT28 primers and gene amplification is carried out for 30 cycles at the following temperatures (96°C, 15 sec; 54°C, 15 sec; 72 0
C,
30 sec). The fragement is then digested with restriction enzymes BamHI/HindIII and cloned into vector pRIT28 which has been digested with the same enzymes, giving: pRIT28G2sub. The nucleotide sequence of G2sub is determined by DNA sequencing on an ABI automated sequencing appliance in accordance with the manufacturer's (Applied Biosystems) recommendations. The G2sub fragment is digested with BamHI and HindIII and cloned into shuttle vector pSE'G2subBBXM (7666bp) (FIGURE 3).
III) Construction of G2del: (see FIGURE In the same way, the G2 gene fragment from which the part encompassing the 4 phenylalanine residues, from amino acid 162 to amino acid 170, has been deleted is generated by PCR in the form of two fragments: upstream 15 using primers RIT27/TH48 on the one hand and downstream using RIT28/TH11 on the other. The primers TH11 and TH48 -are complementary to each other over 13 nucleotides.
TH11 5'-GTGCCGAGCA GCATCTGCAG CA-3' 3'-GGCTTGTTT GGCTTGTTG CACGGCTCGT CGT-5' TH48 Five cycles of the following temperatures (960C, sec; 52 0 C, 1 min; 72 0 C, 1 min) are followed by twenty cycles of the following temperatures (960C, 15 sec; 60 0
C,
sec; 720C, 15 sec). The two amplified fragments are mixed in a single tube and diluted in PCR buffer without primer, and the extension reaction is carried out in five cycles of the following temperatures (96°C, 15 sec; 42 0
C,
sec; 72°C, 1 min). The extension product is diluted 1/100 in PCR buffer containing the RIT27 and RIT28 primers, and gene amplification is effected in 30 cycles of the following temperatures (96 0 C, 15 sec; 60 0
C,
sec; 72°C, 30 sec). The fragment is then digested with restriction enzymes BamHI/HindIII and cloned into vector pRIT28 which has been digested with the same enzymes, giving pRIT28G2del. The nucleotide sequence of G2del is determined by DNA sequencing on an automated ABI sequencing appliance in accordance with the manufacturer's (Applied Biosystems) recommendations. The G2del fragment is digested with BamHI and HindIII and cloned into shuttle vector pSE'G2delBBXM (7639bp) (FIGURE 3).
IV) Construction of the shuttle vector: An oligonucleotide linker TTCCGCCATG GCTCGAG-3', together with the complementary strand) is inserted into the HindIII site of the plasmid pSZZmpl8XM (Hansson et al., 1992, J. Bacteriol 174: 4239- 4245), thereby creating two additional restriction sites, NcoI and XhoI, downstream of the HindIII site of the resulting vector pSZZmpl8(XhoI)XM. A gene fragment encoding 198 amino acids, termed BB, from the serum albumin binding region of streptococcal protein G (Nygren et al., 1988, J.Mol.Reconig., 1:69-74), is generated'by 16 PCR, using primers (1 5'-CCGAATTCAA GCTTAGATGC TCTAGCA- AAA GCCAAG-3' and 2 5'-CCCCTGCAGT TAGGATCCCT CGAGAGGTAA -TGCAGCTAAA ATTTCATC-3'), on a template consisting of plasmid pSPG1 (Guss et al., 1986, EMBO 5: 1567-1575).
The fragment is digested with HindIII and XhoI and cloned downstream of the mpl8 multiple cloning site of vector pSZZmpl8(XhoI)XM; the resulting vector, pSZZmpl8(XhoI)BBXM, is digested with NotI and HindIII.
The fragment encompassing ZZ is replaced with another fragment, which has been digested with the same restriction enzymes, from vector pE'mpl8 (Sophia Hober, unpublished). The resulting shuttle vector is termed pSE'mpl8BBXM (FIGURE 3).
EXAMPLE 2: Extraction and analysis of the membrane proteins from recombinant S. xylosus strains: 250 ml of medium (7.5 g of TSB, 12.5 g of yeast extract) containing chloramphenicol (20 gg/ml), together with 5 ml of an overnight preliminary culture of S. xylosus which has been transformed with one of the shuttle vectors pSE'G2BBXM, pSE'G2subBBXM or pSE'G2delBBXM in accordance with the protocol of G6tz et al., 1981, J Bacteriol., 145:74-81, are inoculated into a 1 litre Erlenmeyer flask. The flask is incubated at a temperature of 32 0 C for 6 hours with shaking. The medium is centrifuged at 5000 rpm for 12 min at a temperature of 4°C. The bacterial pellet is resuspended in 40 ml of TST, and 200 ~l of a solution containing lysostaphin (1 mg/ml) are added, followed by 200 ~i of lysozyme (50 mg/ml). The mixture is incubated at a temperature of 370C for one hour with gentle shaking. The solution is then sonicated for 2 min using a Vibra cell appliance which is equipped with a probe whose power is adjusted to 7. The mixture is centrifuged at 13,500 rpm for 20 min at a temperature of 4 0 C. The proteins are affinity-purified: the supernatant is passed through an HSA-Sepharose (human serum albumin) affinity column. After the column has been rinsed, the proteins are eluted with an acid buffer, pH 2.7, and 17 lyophilized.
Theproteins are separated on two identical 12% -SDS-PAGE gels using prestained standard molecular weight markers (Gibco BRL). One gel is stained with Coomassie blue. The second is transferred to a Problot T M (Applied Biosystem) membrane for immunoblotting with anti-Gl specific antibody (obtained from the serum of a rabbit immunized with Gl(aa174-187) peptide in accordance with current immunization protocols). See Figure EXAMPLE 3: Flow cytometry (FACScan T M analysis of the recombinant proteins at the surface of S. xylosus: Cultures of recombinant S. xylosus bacteria are prepared as previously described. In order to make a stock solution, the bacterium is resuspended in a solution of PBS containing 0.1% sodium azide at a final concentration of unity as determined by optical density (600 nm). 30 ul of stock solution are aliquoted into each conical well of a microtitre plate, which is centrifuged at 550 g for 10 minutes at 4°C. The bacterial pellet is resuspended in a volume of 150 Al of PBS solution containing 200-times diluted anti-G2 polyclonal rabbit serum (titred 1/1 280 000), and the mixture is incubated for 30 minutes. The bacterial cells are rinsed twice with PBS and incubated in 150 tl of a PBS solution containing 100-times diluted FITC anti-rabbit (Sigma) for minutes. After the cells have been rinsed twice with PBS buffer, they are resuspended in a Falcon tube containing 1 ml of 1% PBS-paraformaldehyde buffer.
The samples which have thus been prepared are analysed in a FACScanTM (Becton Dickinson) apparatus. The fluorescence distribution of each cell suspension is analysed using LYSIS IITM software and is depicted by the fluorescence histograms. See Figure 4.
EXAMPLE 4: Modulation from secretion/insertion to secretion: Using different shuttle vectors, it is possible
I
18 to insert termination codons upstream of the membrane anchorage-encoding region, XM, as shown in Figure 6. A -unique XhoI restriction site between BB and XM was used to insert a double-stranded oligonucleotide which encodes three termination codons (Ter) in both orientations, while introducing an Aat II restriction site: Aat II Ter Ter Ter--> GAC GTC TAA TGA TAA TTA TCA TTA G-3' 3'-G CAG ATT ACT ATT AAT AGT AAT CAG Ter Ter Ter Thus, vectors pSE'G2subBBXM and pSE'G2delBBXM are digested with Xho I and ligated to the double-stranded oligonucleotide, which has previously been 5' phosphorylated. The resulting vectors are, respectively, pSE'G2subBB[Ter]XM (7693bp) and pSE'G2delBB[Ter]Xm (7666bp). Proteins G2subBB and G2delBB can be obtained by culturing E. coli or S. xylosus cells which have been transformed, respectively, with these two vectors and then purifying on an affinity column (HSA-sepharose).
Figure 7 shows: in A) the SDS-PAGE gel; separation, under reducing conditions, of the proteins secreted from S. xylosus, with 1 and 2 representing proteins G2subBB and G2delBB, respectively, of the expected sizes: 35.23 Kda and 34.28 Kda; in B) the immunoblot of the proteins demonstrates that the antibody which is specific for the G(aa174-187) region or G1 of RSV readily recognizes the two secreted proteins. Very little proteolytic degradation was observed. In addition, FACSCAN analysis was carried out on the different S. xylosus strains using rabbit anti-BB polyclonal antibody. Figure 8 shows that the spectra for S. xylosus harbouring shuttle vectors pSE'mpl8BBXM, pSE'G2subBBXM and pSE'G2delBBXM are displaced in their axis of fluorescence intensity towards the right, that is towards the presence of heterologous antigens at the bacterial surface. By contrast, the spectra for S. xylosus harbouring shuttle vectors pSE'G2subBBTerXM and pSE'G2delBBTerXM 19 are not displaced, indicating that heterologous antigens are absent from the bacterial surface and that they were -found in, and purified from, the culture medium.
19 bis SEQUENCE LISTING GENERAL INFORMATION:
APPLICANT:
NAME: PIERRE FABRE MEDICAMENT STREET: 45, PLACE ABEL GANCE CITY: BOULOGNE COUNTRY: FRANCE POSTAL CODE (ZIP): 92100 (ii) TITLE OF INVENTION: PRODUCTION OF PEPTIDES WHICH ARE ANALOGUES OF HYDROPHOBIC PEPTIDES RECOMBINANT PEPTIDE, CORRESPONDING DNA SEQUENCE (iii) NUMBER OF SEQUENCES: 6 (iv) COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (EPO) (vi) PRIOR APPLICATION DATA APPLICATION NUMBER FR 9413307 FILING DATE 07-NOV-1994 INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 303 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (ix) FEATURE NAME/KEY CDS (B)LOCATION 1..303 20 SEQUENCE LIST INFORMATION-FOR SEQ ID N0-:1 TYPE =amino acids and nucleotides LENGTH =101 amino acids, 303 nucleotides STRANflEDNESS single TOPOLOGY linear MOLECULE TYPE protein 130 N -Thr Val Lys Thr Lys Asn Thr Thr Thr Thr Gin Thr Gin GTG AAA ACC AAA AAC ACC ACG ACC ACC CAG ACC CAG 143 Pro Ser Lys Pro Thr Thr Lys Gln Arg Gin Asn Lys Pro Pro Asn Lys Pro Asn CCG AGC AAA CCG ACC ACC AAA CAG CGT CAG AAC AAA CCG CCG AAC AAA CCG AAC 161 Asn Asp Phe AAC GAT ri-C His Phe Giu Vai Phe CAT TTC GAA GTG TTC Asn Phe Val Pro Cys AAC TTC GTG COG TGC 174 Ser
ACC
Ile Cys Ser Asn ATC TGC AGC AAC 179 Asn AA C 197 Lys
AAA
187 Pro Thr Cys Trp Ala Ile Cys Lys Arg Ile Pro Asn Lys Lys Pro Gly Lys CCG ACC TGC TGG GCG ATO TGC AAA CGT ATC CCG AAC AAA AAA CCG GGC AAA Thr Thr Thr Lys Pro Thr Lys Lys Pro Thr Phe Lys Thr ACC ACG ACC AAA COG ACC AAA AAA CCG ACC TTC AAA ACC Thr Lys Lys Asp ACC AAA AAA GAT 215 His Lys Pro Gin Thr Thr Lys Pro Lys Glu Val Pro Thr Thr Lys CAT AAA COG CAG ACC ACC AAA COG AAA GAA GTG COG ACC ACC AAA 230 Pro C Ter OCA 3' 21 INFORMATION FOR SEQ ID NO:2 TYPE amino acids and nucleotides -LENGTH 101 amino acids, 303 nucleotides STRANDEDNESS single TOPOLOGY linear MOLECULE TYPE protein 130 -Thr Ala Gin Thr Lys Gly Arg Ile Thr Thr Ser Thr Gin S' -ACC GCG CAG ACC AAA GGC CGT ATC ACC ACC AGC ACC CAG Thr Asn Lys Pro Ser Thr Lys Ser Arg Ser Lys Asn Pro Pro Lys Lys Pro Lys ACE AAC AAA CCG AGC ACE AAA AGC CGT AGC AAA AAC CCC CCG AAA AAA CCG AAA 161 Asp
GAT
Asp Tyr His Phe Glu Vol Phe Asn Phe Vol Pro Cys GAT TAC CAC TTC CAA GTG TTC AAC TTC GTG CCC TGC 174 5cr Ile Cys Gly Asn AGC ATE TGC CCC AAC 179 187 Asn Gin Leu Cys Lys 5cr Ilie Cys Lys Thr Ile AAC CAG CTG TGC AAA AGC ATC TGC AAA ACC ATC 197 Lys
AAA
Pro Ser Asn Lys Pro Lys Lys CCG AGE AAC AAA CCG AAA AAG Thr Lys Thr Thr Asn Lys Arg ACC AAA ACE ACC AAC AAA CT Pro Thr Ilie Lys Pro Thr Asn Lys Pro Thr CCC ACC ATC AAA CCC ACC AAC AAA CCG ACC 213 230 Asp Pro Lys Thr Pro Ala Lys Met Pro Lys Lys iu Ile Ile Thr Asn -C Ter CAT CCC AAA ACC CCG GC AAA ATO CCC AAC AAC CAA ATC ATC ACC AAC -3' 22 INFORMATION FOR SEQ ID NO:3 TYPE amino acids and nucleotides -LENGTH 101 amino acids, 303 flucleotides STRANDEDNESS single TOPOLOGY linear MOLECULE TYPE protein 130 -Thr Vol Lys Thr Lys Asn Thr Thr Thr Thr Gin Thr Gin GTG AAA ACC AAA AAC ACC ACG ACC ACC CAG ACC CAG 143 Pro CCc; 161 Asn
AAC
Ser Lys Pro Thr Thr Lys Gin Arg Gin Asn Lys Pro Pro Asn Lys Pro Asn AGC AAA CCG ACC ACC AAA CAG CGT CAG AAC AAA CCG CCG AAC AAA CCG AAC Asp Phe His Phe Giu Vol Phe Asn Phe Vol Pro GAT TTC CAT TIC GAA GTG TIC AAC TIC GTG CCG Ser Ile Cys Ser Asn AGC ATC TGC AGC AAC 179 187 Asn Pro Thr Cys Trp Alo Ile Ser Lys Arg Ile Pro Asn Lys Lys Pro Gly Lys AAC CCG ACC ICC TGG GCG ATC AGC AAA CGT AIC CCG AAC AAA AAA CCG CCC AAA 197 Lys Thr Ihr Thr Lys Pro Thr Lys Lys Pro Thr Phe Lys Thr AAA ACC ACG ACC AAA CCG ACC AAA AAA CCG ACC TIC AAA ACC Thr Lys Lys Asp ACC AAA AAA GAT 215 His Lys Pro CAT AAA CCG Gin Thr Thr CAC ACC ACC Lys Pro AAA CCG Lys Clu Vol Pro Thr Thr Lys AAA CAA CTC CCC ACC ACC AAA 230 Pro C Ter CCA 3' 23 INFORMATION FOR SEQ ID NO:4 TYPE amino acids and nucleotides -LENGTH 101 amino acids, 303 nucleotides STRANDEDNESS single TOPOLOGY =linear MOLECULE TYPE protein 130 N -Thr Ala
GCG
Gin Thr Lys Gly Arg Ile Thr Thr Ser Thr Gin CAG ACC AAA CCC CCT ATC ACC ACC ACC ACC CAC 143 Thr Asn Lys Pro Ser Thr Lys Ser Arg Ser ACC AAC AAA CCC AGC ACC AAA ACC CGT AC Lys Asn Pro AAA AAC CCG Pro Lys Lys Pro Lys CCG AAA AAA CCC AAA 161 Asp
CAT
179 Asn
AAC
197 Lys
AAA
Asp Tyr His Phe Ciu Vai Phe Asn Phe Val Pro Ser CAT TAC CAC TTC CAA CTC TTC AAC TTC CTC CCC AC 174 Ser
AGC
Ile Cys Ciy Asn ATC TCC CCC AAC Gin Leu Cys Lys Ser Ilie Ser Lys Thr Ile Pro Ser Asn Lys Pro Lys Lys CAC CTC TCC AAA ACC ATC ACC AAA ACC ATC CCC ACC AAC AAA CCC AAA AAG Pro Thr Ilie Lys Pro CCC ACC ATC AAA CCC Thr Asn Lys Pro Thr Thr ACC AAC AAA CCC ACC ACC Lys Thr Thr Asn Lys Arg AAA ACC ACC AAC AAA CCT 215 Asp Pro Lys CAT CCC AAA Thr Pro Ala Lys Met ACC CCC CC AAA ATC Pro Lys Lys Ciu Ile Ilie Thr CCG AAC AAG CAA ATC ATC ACC 230 Asn C Ter AAC 3' 24 INFORMATION FOR SEQ ID TYPE amine acids -LENGTH 30 amino acids STRANDEDNESS single TOPOLOGY =linear MOLECULE TYPE protein 160 162 163 165 168 170 173 Asn Asn Asp Ser His Ser Glu Val Ser Asn Ser Vol Pro Ser Ser 175 176 182 186 Ile Cys Ser Asn Asn Pro Thr Cys Trp Ala Ile Ser Lys Arg Ile 25 INFORMATION FOR SEQ ID NO:6 TYPE amine acids and nucleotides -LENGTH 30 amino acids STRANDEDNESS single TOPOLOGY linear MOLECULE TYPE protein 160 Asn Asn Val Pro Ser Ser Ile Cys Ser Asn Asn Pro Thr Cys Trp 175 Ala Ile Ser Lys Arg Ile Pro Asn Lys Lys Pro Gly Lys Lys Thr FIGURE LEGENDS Figure 5: A) Staining with Comassie blue: SDS Page gel of fusion proteins which were extracted from the membranes of bacteria harbouring different constructs and purified on an albumin affinity column: Well HW: Molecular size markers (in Kda).
well 1: S. xylosus [pSE'G2BBXM].
well 2: S. xylosus [pSE'G2subBBXM].
well 3: S. xylosus [pSE'G2delBBXM].
B) Immunoblot, obtained with rabbit anti-Gl polyclonal antibody, of the fusion proteins which were extracted from the membranes of bacteria harbouring different constructs and purified on an albumin affinity column.
Well HW: Prestained molecular size markers (in Kda).
well 1: S. xylosus [pSE'G2BBXM].
well 2: S. xylosus [pSE'G2subBBXM].
well 3: S. xylosus [pSE'G2delBBXM].
Figure 7: A) Staining with Comassie blue: SDS-page gel of fusion proteins which were secreted from bacteria harbouring different constructs and purified on an albumin affinity column: well 1: xylosus [pSE'G2delBB] (34.28 Kda).
well 2: S. xylosus [pSE'G2subBB] (35.23 Kda).
well HW: Molecular size markers (in Kda).
26 B) Immunoblot, obtained with rabbit anti-G1 (RSV) polyclonal antibody, of the fusion proteins which were -secreted from bacteria harbouring different constructs and purified on an albumin affinity column.
well 1: S. xylosus [pSE'G2delBB] (34.28 Kda).
well 2: S. xylosus [pSE'G2subBB] (35.23 Kda).
well HW: Prestained molecular size markers (in Kda).
6 6
SO*
0.
S. 5659
S
*0 0*
S
*0 See.
6@ 6
S
@5 The claims defining the invention are as follows: 1. Process for secreting a biologically active recombinant peptide which is an analogue of a natural peptide having at least one hydrophobic region, characterized in that it comprises the steps of: a) introducing into a cell a DNA construct which includes: elements ensuring expression and secretion of the said peptide by the cell, and a sequence encoding a peptide whose amino acid sequence differs from the sequence of the natural peptide by at least one modification in a non-transmembrane hydrophobic region of the peptide leading to the hydrophobicity reduction of the peptide; b) culturing the said cell; and c) recovering the said recombinant peptide and/or the cells harbouring the said recombinant peptide.
15 2. Process according to Claim 1, characterized in that at least one hydrophobic amino acid of the sequence of the natural peptide is replaced by a nonhydrophobic amino acid.
3. Process according to one of Claims 1 and 2, characterized in that at least one hydrophobic amino acid is deleted from the sequence of the natural peptide.
4. Process according to one of Claims 1 to 3, characterized in that the hydrophobic amino acid is selected from the following group: tryptophan, phenylalanine, proline, valine, alanine, isoleucine, leucine and methionine.
Process according to one of Claims 1 to 4, characterized in that the 25 DNA construct includes a secretory signal sequence which is linked operationally to the DNA sequence which encodes the recombinant peptide and ensures translocation of the said peptide and its secretion outside the cell.
6. Process according to one of Claims 1 to 4, characterized in that the DNA construct includes a signal sequence which is linked operationally to the DNA sequence which encodes the said peptide and which allows the peptide to be translocated across the membrane of the host cell and anchored to the membrane.
7. Recombinant peptide which can be obtained by the 7 i- I MAW:PP:#25211.RSI 26 February 1999
Claims (22)
- 8. Recombinant peptide according to Claim 7, charac- terized in that it is anchored to the surface of the host cell.
- 9. Recombinant peptide according to one of Claims 7 or 8, characterized in that it is an analogue of an RSV structural protein or of a fragment of such a protein. Recombinant peptide according to one of Claims 7 to 9, characterized in that it comprises a sequence which is an analogue of protein G of RSV, subgroup A or B.
- 11. Recombinant peptide according to one of Claims 7 to 10, characterized in that it comprises a sequence which is an analogue of the sequence encompassed between residues 130 and 230 of RSV protein G.
- 12. Recombinant peptide according to one of Claims 7 to 11, characterized in that it exhibits one of the sequences ID No. 1, No. 2, No. 3, No. 4, No. 5 or No. 6.
- 13. Nucleotide sequence which encodes a peptide according to one of Claims 7 to 12.
- 14. Nucleotide sequence according to Claim 13, characterized in that it additionally includes elements which ensure expression of the peptide in one or more specific host cells. Nucleotide sequence according to one of Claims 13 or 14, characterized in that it is a DNA sequence.
- 16. Nucleotide sequence according to one of Claims 13 or 14, characterized in that it is an RNA sequence.
- 17. Expression vector, characterized in that it includes a nucleotide sequence according to one of Claims 13 to 16.
- 18. Pharmaceutical composition which is intended to be administered to a mammal in order to bring about the in-situ production of a peptide, characterized in that it contains an expression vector according to Claim 17.
- 19. DNA sequence which can be used in the process according to one of Claims 1 to 6, characterized in that it includes: a functional secretory signal sequence, a DNA sequence which encodes a recombinant peptide which is an analogue of a natural peptide, with the recombinant peptide sequence exhibiting at least one modification in a non-transmembrane hydrophobic region of the natural peptide. Recombinant cell, characterized in that it harbours a DNA sequence according to one of Claims 15 and 16 or 19.
- 21. Cell according to Claim 20, characterized in that it is a gram-negative bacteria.
- 22. Cell according to Claim 20, characterized in that it is a gram-positive bacteria.
- 23. Cell according to Claim 20, characterized in that it is a yeast cell.
- 24. Cell according to Claim 20, characterized in that it is a mammalian :76. 0 C '00, so C eqCo C C C 0* C. cell. is selected carnosus. Bacterium according to one of Claims 22 or 23, characterized in that it from Escherichia coli, Staphylococcus xylosus, Staphylococcus
- 26. Gram-negative bacterium according to Claim 21, characterized in that the recombinant DNA sequence is integrated into the chromosome of the host.
- 27. Gram-positive bacterium according to Claim 22, characterized in that it harbours a recombinant DNA sequence which is integrated into the chromosome of the host.
- 28. Pharmaceutical composition, characterized in that it contains a cell 25 according to one of Claims 20 to 27.
- 29. A process according to any one of Claims 1 to 6 substantially as hereinbefore described. Recombinant peptide according to any one of claims 7 to 12 substantially as hereinbefore described.
- 31. An expression vector according to claim 17 substantially as Shereinbefore described. ii MAW:PP:#25211.RS1 26 February 1999
- 32. A pharmaceutical composition according to claim 18 or 28 substantially as hereinbefore described.
- 33. A cell according to any one of claims 20 to 24 substantially as hereinbefore described.
- 34. A bacterium according to any one of claims 25 to 27 substantially as hereinbefore described. DATED: 26 February 1999 CARTER SMITH BEADLE Patent Attorneys for the Applicants: PIERRE FABRE MEDICAMENT woo* *wee N ow a 09 0 0 09 .00. 0 00 G o 0OS 9. 9 9 S S. i ~I -o MAW:PP:#25211.RS1 26 February 1999 PATENT PRODUCTION OF PEPTIDES WHICH ARE ANALOGUES OF HYDROPHOBIC PEPTIDES, RECOMBINANT PEPTIDE, CORRESPONDING DNA SEQUENCE Applicant: PIERRE FABRE MEDICAMENT ABSTRACT The present invention relates to a process for secreting a biologically active recombinant peptide which is an analogue of a natural peptide having at least one hydrophobic region, characterized in that cells which are transformed with a nucleic acid construct which includes elements ensuring expression and secretion of the said peptide by the cell, and a sequence encoding a peptide whose amino acid sequence differs from the sequence of the natural peptide by at least one modification in a non-transmembrane hydrophobic region of the peptide, are cultured and in that the peptide and/or the cells harbouring the said recombinant peptide are recovered. The invention also relates to a recombinant peptide which can be obtained in this manner, to a corresponding DNA sequence and to a bacterium which harbours it.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR9413307A FR2726576B1 (en) | 1994-11-07 | 1994-11-07 | PRODUCTION OF HYDROPHOBIC PEPTIDE-LIKE PEPTIDES, RECOMBINANT PEPTIDE, CORRESPONDING DNA SEQUENCE |
| FR94/13307 | 1994-11-07 | ||
| PCT/FR1995/001464 WO1996014409A1 (en) | 1994-11-07 | 1995-11-07 | Production of recombinant peptides as natural hydrophobic peptide analogues |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| AU4120096A AU4120096A (en) | 1996-05-31 |
| AU704594B2 true AU704594B2 (en) | 1999-04-29 |
Family
ID=9468565
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU41200/96A Ceased AU704594B2 (en) | 1994-11-07 | 1995-11-07 | Production of recombinant peptides as natural hydrophobic peptide analogues |
Country Status (8)
| Country | Link |
|---|---|
| EP (1) | EP0791059A1 (en) |
| JP (1) | JPH10508479A (en) |
| AU (1) | AU704594B2 (en) |
| CA (1) | CA2204511A1 (en) |
| FR (1) | FR2726576B1 (en) |
| NZ (1) | NZ296562A (en) |
| WO (1) | WO1996014409A1 (en) |
| ZA (1) | ZA959417B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6994857B2 (en) | 2000-04-12 | 2006-02-07 | Human Genome Sciences, Inc. | Albumin fusion proteins |
| US7045318B2 (en) | 1995-12-30 | 2006-05-16 | Delta Biotechnology Limited | Recombinant fusion proteins to growth hormone and serum albumin |
| US7507413B2 (en) | 2001-04-12 | 2009-03-24 | Human Genome Sciences, Inc. | Albumin fusion proteins |
| US7847079B2 (en) | 2001-12-21 | 2010-12-07 | Human Genome Sciences, Inc. | Albumin fusion proteins |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5789653A (en) * | 1995-01-10 | 1998-08-04 | University Of Edinburgh | Secretory gene trap |
| AUPO026596A0 (en) * | 1996-06-05 | 1996-06-27 | Biomolecular Research Institute Limited | Viral peptide |
| FR2766192B1 (en) * | 1997-07-17 | 2001-07-13 | Pf Medicament | EPITOPES OF RSV AND ANTIBODIES COMPRISING SAME, USEFUL IN DIAGNOSIS AND THERAPY |
| FR2819810B1 (en) * | 2001-01-23 | 2004-05-28 | Pf Medicament | NON-GLYCOSYL PEPTIDES DERIVED FROM RSV PROTEIN G AND THEIR USE IN A VACCINE |
| US10066240B2 (en) * | 2014-02-14 | 2018-09-04 | Symvivo Corporation | Nucleic acid vectors and uses thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1987004728A1 (en) * | 1986-02-03 | 1987-08-13 | Cambridge Bioscience Corporation | Peptides for the diagnosis of htlv-iii antibodies, their preparation and use |
| GB2188639A (en) * | 1986-04-04 | 1987-10-07 | Hoffmann La Roche | Expression and purification of an HTLV-111 GAG/ENV gene protein |
| EP0249477A2 (en) * | 1986-06-12 | 1987-12-16 | Immunex Corporation | Functional recombinant analog polypeptides devoid of hydrophobic amino acids |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH01501357A (en) * | 1986-01-14 | 1989-05-18 | ユニバ−シテイ・オブ・ノ−ス・カロライナ | Vaccines for human respiratory viruses |
| US5223254A (en) * | 1987-09-29 | 1993-06-29 | Praxis Biologics, Inc. | Respiratory syncytial virus: vaccines |
| ES2176174T3 (en) * | 1988-12-22 | 2002-12-01 | Genentech Inc | PROCEDURE FOR PREPARATION OF WATER SOLUBLE POLYPEPTIDES. |
-
1994
- 1994-11-07 FR FR9413307A patent/FR2726576B1/en not_active Expired - Fee Related
-
1995
- 1995-11-07 ZA ZA959417A patent/ZA959417B/en unknown
- 1995-11-07 WO PCT/FR1995/001464 patent/WO1996014409A1/en not_active Application Discontinuation
- 1995-11-07 CA CA002204511A patent/CA2204511A1/en not_active Abandoned
- 1995-11-07 AU AU41200/96A patent/AU704594B2/en not_active Ceased
- 1995-11-07 JP JP8515108A patent/JPH10508479A/en active Pending
- 1995-11-07 EP EP95939336A patent/EP0791059A1/en not_active Withdrawn
- 1995-11-07 NZ NZ296562A patent/NZ296562A/en unknown
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1987004728A1 (en) * | 1986-02-03 | 1987-08-13 | Cambridge Bioscience Corporation | Peptides for the diagnosis of htlv-iii antibodies, their preparation and use |
| GB2188639A (en) * | 1986-04-04 | 1987-10-07 | Hoffmann La Roche | Expression and purification of an HTLV-111 GAG/ENV gene protein |
| EP0249477A2 (en) * | 1986-06-12 | 1987-12-16 | Immunex Corporation | Functional recombinant analog polypeptides devoid of hydrophobic amino acids |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7045318B2 (en) | 1995-12-30 | 2006-05-16 | Delta Biotechnology Limited | Recombinant fusion proteins to growth hormone and serum albumin |
| US7550432B2 (en) | 1995-12-30 | 2009-06-23 | Novozymes Biopharma Uk Limited | Recombinant fusion proteins to growth hormone and serum albumin |
| US6994857B2 (en) | 2000-04-12 | 2006-02-07 | Human Genome Sciences, Inc. | Albumin fusion proteins |
| US7482013B2 (en) | 2000-04-12 | 2009-01-27 | Human Genome Sciences, Inc. | Albumin fusion proteins |
| US7507413B2 (en) | 2001-04-12 | 2009-03-24 | Human Genome Sciences, Inc. | Albumin fusion proteins |
| US7847079B2 (en) | 2001-12-21 | 2010-12-07 | Human Genome Sciences, Inc. | Albumin fusion proteins |
| US8071539B2 (en) | 2001-12-21 | 2011-12-06 | Human Genome Sciences, Inc. | Albumin fusion proteins |
| US8252739B2 (en) | 2001-12-21 | 2012-08-28 | Human Genome Sciences, Inc. | Albumin fusion proteins |
| US8513189B2 (en) | 2001-12-21 | 2013-08-20 | Human Genome Sciences, Inc. | Albumin fusion proteins |
| 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CA2204511A1 (en) | 1996-05-17 |
| ZA959417B (en) | 1996-06-11 |
| WO1996014409A1 (en) | 1996-05-17 |
| EP0791059A1 (en) | 1997-08-27 |
| JPH10508479A (en) | 1998-08-25 |
| FR2726576A1 (en) | 1996-05-10 |
| FR2726576B1 (en) | 1997-01-31 |
| AU4120096A (en) | 1996-05-31 |
| NZ296562A (en) | 1999-11-29 |
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| MK14 | Patent ceased section 143(a) (annual fees not paid) or expired |