AU614174B2 - The expression of homologous and heterologous proteins, which are able to function, on the outer membrane of e. coli and other gram-negative bacteria - Google Patents
The expression of homologous and heterologous proteins, which are able to function, on the outer membrane of e. coli and other gram-negative bacteria Download PDFInfo
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- AU614174B2 AU614174B2 AU40160/89A AU4016089A AU614174B2 AU 614174 B2 AU614174 B2 AU 614174B2 AU 40160/89 A AU40160/89 A AU 40160/89A AU 4016089 A AU4016089 A AU 4016089A AU 614174 B2 AU614174 B2 AU 614174B2
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- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16111—Influenzavirus A, i.e. influenza A virus
- C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
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Description
BEHRINGWERKE AKTIENGESELLSCHAFT 88/B 023 Ma 674 Dr. Lp/Fa./rd The expression of homologous and heterologous proteins, which are able to function, on the outer membrane of E.coli and other Gram-negative bacteria The invention relates to fusion proteins with the outer membrane protein A (OmpA) of Gram-negative bacteria and to a process for the expression of homologous and heterologous proteins on the outer membrane of E.coli and other Gram-negative bacteria. This entails the proteins which are to be expressed being inserted in the form of their cDNA into the outer membrane protein A (OmpA) in the region of the codons of one of the four outer apexes of this protein. It is furthermore possible for these proteins there additionally to be inserted into a "stalk" which is formed from the amino terminus and carboxy terminus of the hemagglutinin molecule from influenza virus and in turn is inserted into one of the OmpA apexes. The fusion proteins are expressed in the bacteria in a controlled manner.
**It is additionally possible, in order to improve the isolation of the proteins of interest, to incorporate in each case flanking protease- or collagenase-sensitive sequences.
OmpA is among the principal membrane proteins of E.coli and other Gram-negative bacteria such as Shigella dysenteriae and whereas no function is as yet known serves some bacteriophages and colicins as receptor structure for infection and exerting a toxic effect, respectively.
A model has been proposed, from the protein sequence (R.
Chen et al. (1980), Proc. Natl. Acad. Sci., USA, 77, 4592 4596) and from the occurrence of mutations to phage resistance predominantly at positions near the amino acids (AA) 25, 70, 110 and 154 of E.coli OmpA, according to which OmpA passes eight times through the cell membrane, and the abovementioned positions represent the i ~I 2 four apexes of the "outside" loops. The AA numbering used hereinafter is that of MORONA et al. Morona et al. (1985) J. Bacteriology 164, 539 543), which takes the mature E.coli protein without a leader peptide as the basis.
FREUNDL et al. Freundl et al. (1986) J. Mol. Biol.
188, 491 494) have incorporated short peptides of about AA encoded by adapter and linker sequences into positions 154 and 162 of E.coli OmpA in order to demonstrate that the insertion at 154 is sensitive to proteinase K, and is thus accessible from outside, whereas the insertion at 162 was not cleaved because it is in the transmembrane region. Finally, it is suggested in this paper that it is possible for short peptides to be brought to the cell surface by insertions, although the particular length and AA sequence ought to limit greatly the suitability of such peptides.
By contrast, the present invention shows that smaller protein segments, such as antigenic determinants (with a S..*20 length of about 30 AA), but also complete proteins such as, for example, the hemagglutinin (HA) of influenza virus A, the Staphylococcus aureus a toxin, the restriction endonuclease EcoRI or the lacZ gene product (9galactosidase), can, by insertion into the region of one 25 of the abovementioned four positions, preferably in apex position 3 or 4 (AA 110 or 154 of E.coli OmpA or of Shigella dysenteriae OmpA), be expressed on the bacterial membrane and transported through to the outside. In a preferred embodiment, the insertion is additionally carried out in such a way that the "stalk region" of the said hemagglutinin, specifically its ascending limb from about AA 1 to about AA 50 and its C-terminal basic structure with about 235 AA encircle the foreign protein as "inner sleeve", while it itself is inserted into the OmpA protein. It is possible in suitable cases, by incorporating a flanking protease- or collagenase cleavage sequence between the carrier of OmpA or OmpA/HA j I I 3 and the fusion protein, for the protein insert to be cleaved out and purified. The insertion of homologous or heterologous proteins into OmpA can furthermore be undertaken with OmpA or OmpA' strains of E.coli and transferred as plasmid expression system into other Gramnegative bacteria such as, in particular, S.typhi or S.typhimurium.
Since the inserted proteins are transported during translation through the membrane to the outside, it is also possible in the manner described above to synthesize proteins with a complicated tertiary structure in the correct folding. Furthermore, it is also possible in this way to bring about the expression of proteins which in normal genetically engineered expression in the cytoplasm damage the host or are incompatible with the ease host cell. The expression of the fusion proteins takes see* place in the host cells in a controlled manner with a suitable inducer.
Cells which express suitable proteins (for example hemagglutinins of influenza viruses or the protein VP1 of foot and mouth disease viruses) in the described manner .0 outside on the cell membrane can be used as oral live s" vaccine.
Consequently, the invention relates to 0* fusion proteins with OmpA, preferably as insertion of a protein or peptide of interest into a region of the four outer apexes of OmpA, particularly preferred in turn being the region of the third apex (position AA 110 in the OmpA from E.coli or AA 115 in the OmpA from S.dysenteriae); it is possible, in order to stabilize the structure on the surface of E.coli or other Gram-negative bacteria, for the insertions to be flanked by the AA from 1 to about of the amino terminus and about 235 AA of the carboxy terminus of the influenza virus 4 hemagglutinin. To improve the isolation it is possible to incorporate another "sleeve" composed of protease- or collagenase-sensitive sequences.
A process for the preparation of the proteins mentioned under and The use of the resulting Gram-negative bacteria with the OmpA fusion proteins on their surface as live vaccine when the non-OmpA constituent is of interest as immunogen.
The invention is further defined in the patent claims and detailed further in the examples which follow.
*too Examples: 1. Synthesis of hemagglutinin of influenza A virus as fusion protein in the third apex (region of position AA 110 or AA 119) of OmpA in E.coli.
Initially, the E.coli OmpA gene was isolated by the method of CHEN et al. (loc. cit.) and inserted into the expression vector pHK236 as follows. Plasmid pHK236 is composed of the origin region of pBR328, 20 of a promoter-terminator ampicillin-resistance segment from pOB3 (E.Amann et al. (1983) Gene 22, 167-178) and of a LacI Q gene fragment from pJF118u Firste et al. (1986) Gene 48, 119-131); Fig.
1 shows the construction of pHK236. For uptake of the OmpA gene, it was opened in the mpl2 polylinker region in the SmaI site and the Pst I site by the appropriate restriction endonucleases. It was possible to ligate the OmpA gene after cutting with EcoRV (=cut at position 104 base-pairs (bp) upstream relative to the start codon ATG) and Pst I (=cut at position 99 bp downstream relative to the stop codon TAA) by standard methods into the expression vector pHK236 which had been opened as above.
In the resulting plasmid pHS51, the OmpA gene with 5 t..
0 6000
S
Si...
an inserted length of 1243 bp is cut off from its original promoter and, in its stead, fused to the strong vector promoter Ptac Amann et al. (1983) loc. cit.) which lies under the repressor control of the lacI gene which is likewise located on the plasmid. Fig. 2a summarizes the steps in the synthesis of pHS51.
The OmpA gene from S.dysenteriae was isolated, sequenced and expressed in E.coli by COLE et al.
Cole et al. (1980) J. Bacteriol. 149, 145 150). The Shigella gene is substantially identical to the E.coli gene but has, inter alia, 19 hydrophilic AA in the apical region 3 (about position 110 of the E.coli protein) in place of 14 AA in the case of the E.coli gene. The S.dysenteriae gene was ligated into pHK 236 in a manner corresponding to that for the OmpA gene from E.coli, resulting in the plasmid pHS53 (Fig. 2b); in addition an OmpA hybrid gene was constructed, with the Shigella AA sequence extending from position 46 to 233, and the complete protein comprising 330 AA. The construction is depicted diagrammatically in Fig. 2c and results in plasmid pHS56.
Finally, the following 18 bp-long polylinker sequence was ligated after cleavage with HinfI and behind AA 119 in pHS56 after opening with endonuclease HinfI 3 "r 6 Hinfl Clal G T GA A T G A T A- 3'C C A C T T AG CT A T- 'Gly Glu Ser Ile Stul Bcll (Hinfl) -G G C CT A C A A T C T G A G 3 -C C GGACTA GTTTA A C T C Gly Leu Ile Lys Ser Glu 00 and the vector plasmid pHS64 was obtained (Fig. 2d).
Thus the plasmids pHS64 and pHS164 (with an improved promoter/operator region, obtained by exchange from the vector plasmid pHK509 which is otherwise identi- '5 cal (DUisterh6ft and Kr6ger; Diploma thesis of A.
'o Disterh6ft, University of Giessen 1987)) are suitable for taking up the hemagglutinin (HA) gene from influenza virus. A hybrid human influenza cDNA serotype H3 hemagglutinin has been described by VERHOEYEN et al. Verhoeyen et al. (1980) Nature 286, 771 776). After cutting out as the HaeIII BamHI (1542 bp) fragment and ligation into the i correspondingly opened StuI/BclI cleavage sites in i the 18 bp polylinker sequence it was possible to obtain the plasmids called pinf4-6 and pinf4-39 (Fig. 3).
The abovementioned hybrid plasmids were used to transform various E.coli strains with a chromosomal OmpA gene (for example 490A, HB101), as well as, for analytical purposes, the strain UH 201.3 (OmpA- S.T. Cole et al. (1982), J. Bacteriol. 149, 145- 150), and the sandwich proteins OmpA/HA/OmpA were
I
r, 1 OOoe 5 *5 o. 2 7 induced by induction with isopropylthiogalactoside (IPTG); however, it is likewise possible to transform, and induce by IPTG, other Gram-negative bacteria. After induction it was possible to detect the OmpA-HA fusion protein in the total protein gels from E.coli, greatly enriched as a principal component among the membrane proteins of the bacterium. In both cases (transformation with pinf4-6 and pinf4-39, respectively), the immune reaction with an HA antibody by immunoblotting (Western blot) demonstrates the cross-reaction with authentic HA for the OmpA/HA fusion band.
It was possible to demonstrate the presence of correctly formed HA on the outside of the transformed and induced cells by iodine labeling using lactoperoxidase, by the preferred trypsin cleavage on the intact bacterial cells, and by the indirect irimunofluorescence test (IFT). This entailed induced cells being incubated in a first step with antiserum against HA and then with fluoresceinlabeled anti-antibodies in a second step. The induced cells (almost 100% viable according to the IFT procedure) fluoresced distinctly whereas noninduced cells were not stained.
2. Expression of a surface domain of foot and mouth disease (FMD) virus, serotype 0 1 K as OmpA fusion protein on the surface of E.coli or S.typhi.
The DNA sequence corresponding to codons 140-162 of the virus surface protein VP1 (Cheung et al. (1983), J. Virol.48, 451-459) was resynthesized and, with ClaI-StuI ends, cloned in OmpA fusion connection of the correspondingly opened vector pHS164. The oligo-nucleotide sequence (pfmdv-1) inserted into pHS164 is as follows: L I 8 Clal (AT) CG ATT GCT GTG CCC AAC TTG AGA GGT GAC CTT- TAA CGA CAC GGG TTG AAC TCT CCA CTG GAA- *0ge
CC..
CC
CAG GTG TTG GCT CAA AAG GTG GCA CGG ACG CTG- GTG CAC AAC CGA GTT TTC CAC CGT GCC TGC GAC- StuI CCT ACC TCA GG GGA TGG AGT CC- The expression of the VP1 domain was detected on the surface of the induced, pfmdv-1-containing cells by IFT as in Example 1.
5
.C*
oo 3. Synthesis of the Saphylococcus aureus fusion protein in the third apex (region AA 110 or 119) of OmpA in E.coli.
a toxin as of position f C C *t C The gene for the S.aureus a toxin has been isolated and sequenced by KEHOE et al. Kehoe et al.
(1983) Infect. Immuno. 41, 1105-1111). It has been cloned by Bhakdi and coworkers in portable form into the plasmid pUC19 as pUC19-aTox and is obtained therefrom, by cleavage with Clal and HinfI, in two partial fragments which, together with one each of an N-terminal and C-terminal bridging oligonucleotide, allow stepwise reconstruction in the fusion frame with the OmpA gene in pHS64 to give the plasmid pHS140.
It was possible to detect, by IFT and by iodine labeling, the a toxin as well as the hemagglutinin on the outside of the E.coli cells transformed with pHS140 in the customary manner.
7 i 9 5..
r fe 0 a
SWO
C
e*C 0 C *i 25 W: V C
SW
4. Increase in the expression of the S.aureus a toxin by additional incorporation into the "stalk region" of the influenza hemagglutinin.
It was possible substantially (about four-fold) to increase the surface expression of the S.aureus a toxin by additionally incorporating this protein into the "stalk region" of the influenza hemagglutinin gene. This entails the codons of AA 1-48 and 279-514 of the amino-terminal and of the carboxy-terminal region, respectively, of the hemagglutinin being ligated into the plasmid pHS164, and the middle "head" region of HA from pinf4-39 (see above) being deleted from AA46 to 281 and reconstructed by an oligonucleotide linker in the said limits. Furthermore, an oligonucleotide linker region in the region which is structurally suitable for this and is located on the protein surface (codon 400-409) is produced by exchange with a second oligonucleotide (plasmid pinf 4-49). The a toxin gene was incorporated into the latter as in Example 2, specifically as the 950 bp NheI-StuI fragment from pHS140. Expression and detections of expression were carried out as in Example 1.
pHS164 and pinf4-49 are detailed in Fig. 4, Fig. Tab. 1 and Tab. 2 as examples of so-called "master plasmids" or vector frameworks. pHS164 contains the OmpA sleeve, and pinf4-49 additionally contains the HA sleeve. Furthermore, an oligonucleotide linker sequence which makes it possible to insert foreign genes is ligated in both "master plasmids".
10 pHS 164 Seq.
Tab. 1 Sequence of pHS 164 (linker in the 3rd domain) tac Promoter 1 51 101 151 201.
*Soo 25 1 boa. 301 351 a boo 6:::.401 451 501 551 601 751 751 100 1051 1101 1151 1201 1251 AATATTCTGA ATACG TGGAATTGTG AGCGGATAAC TTCCAkCATGT GGAATTGTGA TCCTCTGACC AGCCAGAAAA TCTGACCAGC CAGAAAACGA CCGCTCACAA TTCCACATGT ATATTGAGCA GATCCCCCGG GATATTCATG GCGTATTTTG ACAkGCTATCG CGATTACAGT GGCCGCTCCG AALAGATAACAk CCCAGTACCA TGA CA CmTGGT AACCAACTGG GCGCTGGTGC TGGCTTTGAkA ATGGGTTACG GCGTTGAAAA CGGTGCATAC CTGGGTTACC CAATCACTGA TATGGTTTGG CGTGCAGACAk CGATAGGCCT GAT AAATCT -Link er--- GTATTCGCAG GTGGTGTTGA TCTGGAATAC CAGTGGACCA% CTCGTCCGGA CAACG%'C-CTG CAGGGCGAAG CAGCTCCANGT AGTACAGACC AAGC-ACTTCAk ACAAAGCAAC -CCTGAAACCG AGCCAGCTGA GCAACCTGGA TTACAbCCGAC CGCATCGGTT GCCGTGCTCA GTCTGTTGTT TGACAATTAA TCATCGGCTC GTATAATGTG (II Iit It 11,1 if It AA TT TCACA C
GCGGATAACA
C GAT CTTCGT
TCTTCGTCCG
GGAATTCGAG
TGAAGGATTT
GATGATAACG
G GCA C T GG C T C CTGGTACAC TTCATC GACAk
TTTTGGTGGT
ACTGGTTAGG
AAAG CTCAGG
CGACCTGGAC
C CAAAG CTCA
GAGAAAAACC
ATGG-GCCATC
ACAACATCGG
CTGAGCTTGG
AGTTGCTCCG
CTCTGAAGTC
GAAGCG'!CAGG
TCCGAAAGAC
C TGA CGC TTA GATTAC CTGA AGGAAACA GA
ATTTGTGGAA
CCGAGAATTC
AGAATT CCAC
CTCGCCCATC
AAC CGTGTTA AGGC GCAAA
GGTTTCGCTA.'.
TGGTGCTAAA
ACAATGGCCC
TAC CAGGTTA T CGTATG CCG GC GTT CAGTT GTGTACACT C
CAACAATGTG
ACGATACCGG
ACT CCTGAAA TGAC GCACAC GTGTTTC CTA
GCTCCAGCTC
TGACGTTCTG
CTGCTCTGGA
GGTTCCGTAG
CAAC CAGGGT
TCTCCAAAGG
CCATGCGGAA
TTCTGGCGAA
TGG-CGAATCC-
AAATTGTTAT
G GTACA G TTA
TCTCGTTGGA
Start of OmpA
AATGAAAAAG
CCGTAGCGCA
CTGGGCTGG'T
GAC CCATGAA AC C CGTATGT
TACAAAGGCA%
GAC C G CTAAA
GTCTGGGTGG
ACAGGIGAAT
C GTTT CT CCG TCG CTAC CC G
ACCATCGGCA
C CGTTTCGGT
CGGCACCGGA
TTC-VJ.CTTCA
TCAGCTGTAC
TTGTTCTGGG
CTGTCCGAGC
TAT CCC GGCA 11 1301 GACAAGATCT CCGCACGTGG 1351 CACCTGTGAC AACGTGAAAC 1401 CGGATCGTCG CGTAGAGATC End of OMpA 1451 CAGCCGCAGG CTTAAGTTCT 0 0000 0000*0 0 0000 0000 0000 0000 A 00 0 00 0 0 00 00 0 0 00 00 00 00 0 .oo 00 1501 1551 1601 1651 1701 1751 1801 1851 1901 1951 2001 2051 2101 2151 2201 2251 2301 2351 2401 2451 2501 2551 2601 2651
TTTTTTGCCT
AG CAATG CC T
CAGCCTGATA
ATTTGCCTGG
TCAGAAGTGA
GAGAGTAGGG
GAC TGGGC CT TAG GA CAAAT
GAGGGTGGCG
CAGAAGGCCA
GTTTATTTTT
CCCTGATAAA
ACATTTCCGT
TTTTTGCTCA
TTGGGTGCAC
CCTTGAGAGT
AAGTTCTGCT
CAACTCGGTC
ACCAGTCACA
GCAGTGCTGC
ACAACGATCG
GGATCATGTA
TACCAAACGA
TTGCGCAAAC
TTAGTAA.ATT
GCAGCCCAAG
CAGATTAAAT
CGG CAGTAGC AAC GC CGTAG AACTGC CAG'G TTC GTTTTAT
CCGCCGGGAG
GGCAGGACGC
TCCTGACGGA
CTAAATACAT
TGCTT CAATA GTC GC CCTTA
CCCAGAAACG
GAGTGGGTTA
TTTCGCCCCG
ATGTGGCGCG
GCCGCATACA
GAAAAGCATC
CATAAC CATG
GAGGACCGAA
ACTCGCCTTG
CGAGCGTGAC
TATTAACTGG
TATGGGCGAA
AGCGTGCTGC
GAAGTTAAAG
CGTCTGGTAG
GAACTGACTT
CTTCTGTTTT
CAGAACGCAG
GCGGTGGTCC
CGC CGATGGT CAT CAAATAA
CTGTTGTTTG
CGGATTTGAA
CCGCC-ATAAA
TGGCCTTTTT
TCAAATATGT
ATATTGAAAA
TTCCCTTTTT
CTGGTGAAAG
CATCGAACTG
AAGAACGTTT
GTATTATCCC
C TA T TC TCAG
TTACGGATGG
AGTGATAACA
GGAGCTAACC
ATCGTTGGGA
ACCACGATGC
CGAACTACTT
TCCAACCCGG
ACTGATCGAC
GTATCAAAGA
AAAAAC GCTG
TCGTCAGTTA
GGCGGATGAG
AAGCGGTCTG
CACCTGACCC
AGTGTGGGGT
AACGAAAGGC
TCGGTGAAC G
CGTTGCGAAG
CTGCCAGGCA
GC GTTTCTAC
ATCCGCTCAT
AGGAAGAGTA
TGCGGCATTT
TAAAAGATG C
GATCTCAACA
TCCAATGATG
GTGTTGACG C
AATGACTTGG
CATGACAGTA
CTGCGGCCAA
GCTTTTTTGC
ACCGGAGCTG
CTGCNGCAAT
ACTCTAGCTT
TTACTGGCAA
TGCCTGGCTC
CGTTGTAACT
CTGCGGGTTT
TTC CTTACC C
AGAAGATTTT
ATAAAACAGA
CATGC CGAAC
CTCCCCATGC
TCAGTCGAAA
CTCTCCTGAG
CAACGGCCCG
TCAAATTAAG
AAACTCTTTT
GAGACAATAA
TGAGTATTCA
TGCCTTCCTG
TGAAGATCAG
GCGGTAAGAT
AG CA CTTT TA
CGGGCAAGAG
TTGAGTACTC
AGAGAATTAT
CTTACTTCTG
ACAACATGGG
AATGAAG CCA GGCAACAAC G
CCCGGCAACA
r 12 2701 2751 4 2801 2851 K 2901 K 2951 3001 3051 3101 3151 3201 3251 3301 3351 3401 3451 3501 3551 3601 3651 3701 3751 3801 3851 3901.
3951 4001 4051
ATTAATAGAC
C GGC CCTTC C
CGTGGGTCTC
CCGTATCGTA
GAAATAGACA
CTGTCAGACC
TTTTTAATTT
C CAAAATC CC
GAAAAGATCA
CTGCTTGCAA
ATCAAGAGCT
CAGATACCAA
CAAGAACTCT
CAGTGGCTGC
AGACGATAGT
GTGCACACAG
TACAGCGTGA
GACAGGTATC
GCTTC CAGGG AC CTCTGACT
CTATGGAAAA
TGGAGATGGC
TTCACAGTTC
TCCGTTAGCG
CCATGCACCG
CTACAATCCA
GC CGTGACGA
GAGCGATCCT
TGGATGGAGG
GGCTGGCTGG
GCGGTATCAT
GTTAT CTACA
GATCGCTGAG
AAGTT TA CT C
AAAAGGATCT
TTAACGTGAG
AAGGATCTTC
ACAAMAAAAC
ACCAACTCTT
ATACTGTCCT
GTAGCACCGC
TGCCAGTGGC
TAC CGGATAA
CCCAGCTTGG
GCATTGAGAA
CGGTAAGCGG
GGAAACGC CT
TGAGCGTCGA
ACGC CAGCAA
GGACGCGATG
TCCGCAAGAA
AGGTGCCGCC
C GAC GCAACG TGC CAAC CCG
TCAGCGGTCC
TGAAG CTGTC
CGGATAAAGT
TTTATTG CTG
TGCAGCJACTG
CGACGGGGAG
ATAGG TGC CT
ATATATACTT
AGGTGAAGAT
TTTTCGTTCC
TTGAGAT CCT
CACCGCTACC
TTTCCGAAGG
TCTAGTGTAG
CTACATACCT
GATAAGTCGT
GGCGCAGCGG
AGCGAACGAC
AGCGCCACGC
CAGGGTCGGA
GGTATCTTTA
TTTTTGTGAT
CGCGGCCCGA
GATATGTTCT
TTGATTGGCT
GGCTTCCATT
CGGGGAGGCA
TTCCATGTGC
AGTGATCGAA
CCTGATGGTC
TGCAGGACCA
ATAAATCTGG
GGGCCAGATG
TCAGG CAACT
CACTGATTAA
TAGATTGATT
CCTTTTTGAT
ACTGAGCGTC
TTTTTTCTGC
AGCGGTGGTT
TAACTGGC TT C CGTAGTTAG
CGCTCTGCTA
GTCTTACCGG
TCGGGCTGAA
CTACAC CGAA
TTCCCGAAGG
ACAGGAGAGC
TAGTCCTGTC
GCTCGTCAGG
GATGCGCCGC
GCCAAGGGTT
CCAATTCTTG
CAGGTCGAGG
GACAAGGTAT
TCGCCGAGGC
GTTAGGCTGG
GTCATCTAC C
CTTCTGCGCT
AGCCGGTGAG
GTAAGCCCTC
ATGGATGAAC
GCATTGGTAA
TAAAACTTCA
AATCTCATGA
AGACCCCGTA
G CGTAAT CTG
TGTTTGCCGG
CAGCAGAGCG
GC CAC CAC TT
ATCCTGTTAC
GTTGGACTCA
CGGGGGGTTC
CTGAGATAC C
GAGAAAGGCG
GCACGAGGGA
GGGTTTCGCC
GGGGCGGAGC
GTGCGGCTGC
GGTTTGCGCA
GAGTGGTGAA
TGGCCCGGCT
AGGGCGGCGC
GGCATAAATC
TAAGAGCCGC
TGCCTGGACA
13 *fee 0006 0 00 es6* es0 4101 4151 4201 4251 4301 4351 4401.
4451 4501 4551 4601 4651 4701 4751 4801 4851 4901 4951 5001 5051 5101 5151 5201 5251 5301 5351 5401 5451
GCATGGCCTG
ATAATGGGGA
GCCCAGCGCG
TTGCGCTCAC
TTAATGAATC
AGGGTGGTTT
CACCGCCTGG
CCAGCAGGCG
GAGCTGTCTT
GCGCAGCCCG
CGTTGGCAAC
ATGGTTTGTT
TATCGGCTGA
GCAGACGCGC
TGGTGAC CCA
ATGGGAGAAA
ATAAC GC CGG
TCATCC.AGCG
ATTGTGCAC C ACAC CAC CAC
ACAATTTGCG
CAGCAACGAC
A.ATTCAGCTC
ACGTGGCTGG
GG CATACTCT
TGA.ATTGACT
CACCATTCGT
TTGCAAGCTG
CAACG CGGG C
AGGCCATCCA
TCGGCCAGCT
TGCCCGCTTT
GGCCAACGCG
TTCTTTTCAC
CCCTGAGAGA
AAAATC CTGT CGGTATC GT C GACT CGGTAA
CAGCATCGCA
GAAAAC CGGA
ATTTGATTGC
C GAGACAGAA
ATGCGACCAG
ATAATACTGT
AACATTAGTG
GATAGTTAAT
GCCGCTTTAC
GCTGGCACCC
ACGGCGCGTG
TGTTTGCCCG
CGCCATCGCC
CCTGGTTCAC
GCGPACATCGT
CTCTTC CGGG
ATGGTGTCAA
ATCGGAGCTT
ATCCCGATGC
GCCTCGCGTC
TGCAATTCGC
CCAGTCGGGA
CGGGGAGAGG
CAGTGAGACG
GTTGCAGCAA
TTGATGGTGG
GTATCCCACT
TGGCGCGCAT
GTGGGAACGA
CATGGCACT C
GAGTGAGATA
CTTAATGGGC
ATGCTC CAC G
TGATGGGTGT
CAGGCAGCTT
GATCAGCCCA
AGGCTTCGAC
AGTTGATCGG
CAGGGC CAGA
CCAGTTGTTG
GCTTCCACTT
CACGCGGGAA
ATAACGTTAC
CGCTATCATG
CGTAAATGCC
AT CGA CTG CA
CGCCGGAAGC
GCGAACGCCA
GCTAACTTAC
AACCTGTCGT
CGGTTTGCGT
GGCA.ACAGCT
GCGGTCCACG
TTGACGGCGG
ACCGAGATAT
TGCGCCCAGC
TGCCCTCATT
CAGTCGCCTT
TTTATGC CAG C CGC TAACAG CC CAGTC GC G
CTGGTCAGAG
CCACAGCAAT
CTGACGCGTT
GCCGCTTCGT
CGCGAGATTT
CTGGAGGTGG
TGCCACGCGG
TTTCCCGCGT
ACGGTCTGAT
TGGTTTCACA
CCATACCGCG
CGTTGCCCTT
CGGTGCACCA
GAGAAGAAT C
GCAAGACGTA
ATTAATTGCG
GCCAGCTGCA
ATTGGGCGC C
GATTGCCCTT
CTGGTTTGCC
GATATAA CAT C CGCAC CAAC
GCCATCTGAT
CAGCATTTGC
CCCGTTCCGC
C CAGC CAGAC
CGCGATTTGC
TACCGTCTTC
ACATCAAGAA
GGCATCCTGG
GCGCGAGAAG
TCTACCATCG
AATCGCCGCG
CAACGC CAAT
TTGGGAATGT
TTTCGCAGAA
AAGAGACAC C
TTCACCANCCC
AAAGGTTTTG
CGCGCGCGAA
ATGCTTCTGG
14 5501 CGTCAGGCAG CCATCGGAAG CTGTGGTATG GCTGTGCAGG TCGTAAATCA 5551 CTGCATAATT CGTGTCGCTC AAGGCGCACT CCCGTTCTGG ATAATGTTTT 5601 TTGCGCCGACATCATAACGG TTCTGGCAG en 6e 4 4** ~4 a.
S
44445e a 04.~' b 4 545 4* 4* S
*S
4 S 4 44 05 @4 4
*S
SS
S 7 15 pINF4-49 seq.
Tab. 2 Sequence of pinf 4 49 tac ?romoter 1 AATATTCTGA AATGA:GCTGT TGACJATTAAk TCATCGGCTC GTATAATGTG 51 101 1.51 201 251 301.
351 401 4 4511 502.
551 601 651.
701 751 *g*i801 851 :'*.901 5 1 1.001 1.051 1101 11.51
TGGAATTGTG
T TC CA C ATGT T C C T C TGA C C
TCTGACCAGC
CCG GC T CA CA ATATT GAG CA
GATATTCATG
ACA:GCTAkTCG
GGCCGJCTCCG
C C CA GTA CCA A.AC CAACTC-G TGGC-TTT6GAA
GCGTTGAAAA
CTGGGTTACC
TATGGTT.TGG
CGATAGGCCA
CTGGGA CAT C
TGATCAGATT
It I i It 11 1 it it" i ftI II I? AG CGGATAAC
GGAATTGTGA
AG C AGAA CAGAAAAC GA TTC ,-CCATGT GATC CCC CGG GC GTATTTTG
CGATTGCAGT
AAAGATAkACA
TGATACTGGT
GCGCTGGTGC
ATGGGTTACG
CGGTGCA;TAC
CGTG CAGACA%
AGACCTTCCA
ATGCGGTGCC
GAAGT GA CTA AATT-TCACAC AGGAAACAGA CCATGCGGAA GCGGATAAkCA -%TTTGTGG.AA-- TTCTGG-CGAA CGATCTTCGT CCGAGAATTC TGGCGAAkTCC TCTTCGTCCG AGAATTCCAC AAATTGTTAkT GGAATTCGAG CTCGCCCATC GGTAGAGTTA TG.AAGGATTT AACCGTGTTA TCTCGTTGGA Start of OmpA GATGATAACG AGGCGCAkAAA AATGA-AAAAG GGCACTGGCT GGTTTCGCTAk CCGTAGCGCAk CCTGGTLACA:C TGGTGCTAAA CTGGGCTGGT TTCATCAACA AC-AATGGCCC GACCCATGAA TTT.TGGTGGTJ- TACCAGGTTA ACCCGTATGTJ ACTGGTTAGG TCGTAkTGC-CG TACAXAAGGCAk AAAGCTCAGG GCGTTCAGTT GACCGCTAkA CGACCTGGAC GTGTACACTC GTCTGGGTGG Junction :OmpA/HAV C CAAAGCTCA CAAC.AATGTG ACAGGTGAAT GGAAATGACA% ACAGCAPCAGC AACGCTGTGC AAACGG.AACA CTAGTGAAAA CAATCACAGA ATGCTAkCTGA GCTAGTTCAG AGCfljTAGAG AATGGAAGCAk TTCCC-IATGA CAAGCCCmTTr ATATGGAGCA TG%'CCCAAGT ATGTTAkAGCA% CAGGGATGCG GAATGTACCA GAGAAACAAA ATAGCAGGTT TCATAGAAAA TGGTTGGGAG CGGTTTCAGG CAtCAAAATT CTGAGGGC.%C TACTCATATG COTCACTCC-7 -Linker CAkAAACGTAA ACAAGATCAC AAACACCCTG AAGTTGG-CAA CT.AGAGGCCT ATTCGGCGCAk GGAATGATAG ACGG-TTGGTAk 1.201. AGGACAAGCA GCAGATCTTA AAAGCACTCA AGCAGCCATC GACC-%AATCAk
U
4 1 16 1251 1301 ATGGGAAATT GAACAGGGTA ATCGAGAAGA CGAACGAGAA ATTCCATCAA ATCGAAAAGG AATTT-CTAG CGACGTCGAC CCGGGCCAAG AT TCGAGAA t Linker J 1351 ATACGTTGAA GACACTAAA.A TAGATCTCTG GTCTTACAAT GCGGAGCTTC
I
1401 1451 1501 1551 1601 1651 1701 1751 1801 1851 1901 1951 2001 eg 2051 SS** 2101 2151 2201 6* 00 2251 0. 0 2301 TTGTCGCTCT GGAGAATCAA CATACAATTG AACAAGCTGT TTGAAAAAAC AAGGAGGCAA GATGGGCAAT GGTTGCTTCA AAATATACCA TAGAGTCA.AT CAGAAATGGT ACTTATGACC GCATTAAACA ACCGGTTTCA GATCAAAGGT V Jun ction: HA/OmpA CAAAGACTGG ATCAAATCTG AGAAAAACCA TATTCGCAGG TGGTGTTGAA TGGGCCATCA CTGGAATACC AGTGGACCAA CAACATCGGT TCGTCCGGAC AACGGCCTGC TGAGCTTGGG AGGGCGAAGC AGCTCCAGTA GTTGCTCCGG GTACAGACCA AGCACTTCAC TCTGAAGTCT CAAAGCAACC CTGAAACCGG AAGGTCAGGC GCCAGCTGAG CAACCTGGAT CCGAAAGACG TACACCGACC GC.ATCGGTTC TGACGCTTAC CCGTGCTCAG TCTGTTGTTG ATTACCTGAT ACAAGATCTC CGCACGTGGT ATGGGCGAAT ACCTGTGACA ACGTGAA.ACA GCGTGCTGCA GGATCGTCGC GTAGAGATCG AAGTTAAAGG End of OmpA AGCCGCAGGC TTAAGTTCTC GTCTGGTAGA ACCTGACTGA CTCGGAAATC.
CTGAGGGAAA ATGCTGAAGA CAAATGTGAC AACGCTTGCA ATGATGTATA CAGAGACGAA GTTGAACTGA AGTCTGGATA CGATACCGGC GTTTCTCCGG CTCCTGAAAT CGCTACCCGT GACGCACACA CCATCGGCAC TGTTTCCTAC CGTTTCGGTC CTCCAGCTCC GGCACCGGAA GACGTTCTGT TCAACTTCAA TGCTCTGGAT CAGCTGTACA GTTCCGTAGT TGTTCTGGGT AACCAGGGTC TGTCCGAGCG CTCCAAAGGT ATCCCGGCAG CCAACCCGGT TACTGGCAAC CTGATCGACT GCCTGGCTCC TATCAAAGAC GTTGTAACTC AAAACGCTGC TGCGGGTTTT 2351 2401 2451 2501 2551 2601 TTTTTGCCTT TAGTAAATTG AACTGACTTT CGTCAGTTAT GCAATGCCTG CAGCCCAAGC TTCTGTTTTG GCGGATGAGA AGCCTGATAC AGATTAAATC AGAACGCAGA AGCGGTCTGA TTTGCCTGGC -GGCAGTAGCG CGGTGGTCCC ACCTGACCCC CAGAAGTGAA ACGCCGTAGC GCCGATGGTA GTGTGGGGTC AGAGTAGGGA ACTGCCAGGC ATCAAATAAA ACGAAAGGCT
TCCTTACCCA
GAAGATTTTC
TAAAACAGAA
ATGCCGAACT
TCCCCATGCG
CAGTCGAAAG
17 2651 ACTGGGCCTT TCGTTTTATC 2701 2751 2801 2851 2901 2951 3001 3051 3101 3151 3201 3251 3301 3351 3401 3451 3501 3551 3601 *3651 3701 3751 3801 3851
AGGACAAATC
AGGGTGGCGG
AGAAGGC CAT
TTTATTTTTC
C CTGATAAAT
CATTTCCGTG
TTTTGCTCAC
TGGGTGCACG
CTTGAGAGTT
AGTTCTG CTA
AACTCGGTCG
C CAGTCACAG
CAGTGCTGCC
CAACGATCGG
GATCATGTAA
AC CAAAC GAC
TGCGCAAACT
TTAA TA GAC T
GGCCCTTCCG
GTGGGTCTCG
CGTATCGTAG
AAATAGACAG
TGTCAGAC CA
TTTTAATTTA
CGCCGGGAGC
GCAGGACGCC
CCTGACGGAT
TI\JATACATT
GCTTCAATAA
TCGC CCTTAT
CCAGAAACGC
AGTGGGTTAC
TTCGCCC CGA
TGTGGCGCGG
C CGCATACAC
AAAAGCATCT
ATAAC CATGA
AGGACCGAAG
CTCGCCTTGA
GAG CGT GA CA
ATTAACTGGC
GGATGGAGGC
GCTGGCTGGT
CGGTATCATT
TTATCTACAC
ATCGCTGAGA
AGTT TA C TCA
AA.AGGATCTA
TGTTGTTTGT
GGATTTGAAC
C GC CATAAAC
GGCCTTTTTG
CAAATATGTA
TATTGAAAAA
TCCCTTTTTT
TGGTGAAAGT
ATCGAACTGG
AGAAC GTTTT
TATTATCCCG
TATTCTCAGA
TACGGATGGC
GTGATAACAC
GAGCTAACC G
TCGTTGGGAA
C CACGATGC C GAA CTA C TTA
GGATAAAGTT
TTATTGCTGA
GCAGCACTGG
GACGGGGAGT
TAGGTGCCTC
TATATACTTT
GGTGAAGATC
TTTCGTTCCA
TGAGATC CTT
CGGTGAACGC
GTTGCGAAGC
TGC CAGGCAT
CGTTTCTACA
TCCGCTCATG
GGAAGAGTAT
GCGGCATTTT
AAAAGATGC T
ATCTCAACAG
C CAATGATGA
TGTTGACGCC
AT GAC TT GG T
ATGACAGTAA
TGCGGCCAAC
CTTTTTTGCA
C CGGAGC TGA
TTCAGCAATG
CTCTAGCTTC
GCAGGAC CAC TAAItTCTGGA
GGCCAGATGG
CAGGCAACTA
ACTGATTAAG
AGATTGATTT
CTTTTTGATA
CTGAGCGTCA
TTTTTCTGCG
TCTCCTGAGT
AACGGCCCGG
CAAATTAAGC
AACTCTTTTG
AGACAATAAC
GAGTATTCAA
GCCTTCCTGT
GAAGATCAGT
CGGTAAGATC
GCACTTTTAA
GGGCAAGAGC
TGAGTACT CA
GAGAATTATG
TTACTTCTGA
CAACATGGGG
ATGAAGC CAT GCAACAAC GT
CCGGCAACAA
TTCTGCGCTC
GCCGGTGAGC
TAAGCCCTCC
TGGATGAACG
CATTGGTAAC
AAAACTT CAT AT CTCAT GA C
GACCCCGTAG
CGTAAT CTGC 3901 3951
CAAAATCCCT.-TAACGTGAGT
AAAAGATCAA AGGATCTTCT 4001 TGCTTGCAAA CAAAAAAACC ACCGCTACCA GCGGTGGTTT GTTTGCCGGA 18 4051 4101 4151 4201 4251 4301 4351 4401 4451 4501 see 0 4551 000 4601 4651 4701 *09 a 4751 00* 4801 0 4851 4901 4951 so 5001 5051 5101 5151 5201 5251
TCAAGAGCTA
AGATAC CAAA AAGAACT CTG
AGTGGCTGCT
GACGATAGTT
TGCACACAGC
A CAG CGT GAG
ACAGGTATCC
CTTC CAGGGG
CCTCTGACTT
TATGGAAAAA
GGAGATGGCG
TCACAGTTCT
C CGTTAGC GA
CATGCACCGC
TACAATC CAT
CCGTGACGAT
AGCGATCCTT
CATGGCCTGC
TAATGGGGAA
CCCAGCGCGT
TGCGCTCACT
TAATGAATCG
GGGTGGTTTT
ACCGCCTGGC
CCAACTCTTT
TACTGTC CTT
TAGCACCGCC
GCCAGTGGCG
AC CGGATAAG
CCAGCTTGGA
CATTGAGAAA
GGTAAGC GGC GAAACGC CTG GAGC GT CGAT
CGCCAGCAAC
GACGCGATGG
CCGCAAGAAT
GGTGCCGCCG
GACGCAACGC
GCCAACCCGT
CAGCGGTCCA
GAAGCTGTC C AACGCGGG CA GGCCATC CAG
CGGCC.AGCTT
GCCCGCTTTC
GCCAACGCGC
TCTTTTCAC C C CTGAGAGAG
TTCCGAAGGT
CTAGTGTAGC
TA CATAC CTC ATAAGTC GTG
GCGCAGCGGT
GCGAACGACC
GC GC CACG CT
AGGGTCGGAA
GTATCTTTAT
TTTTGTGATG
GCGGCC CGAG
ATATGTTCTG
TGATTGGCTC
GCTTCCATTC
GGGGAGGCAG
TCCATGTGCT
GTGATCGAAG
CTGATGGTCG
TCCCGATGCC
CCTCGCGTCG
GCAATTCGCG
CAGTCGGGAA
GGGGAGAGGC
AGTGAGACGG
TTGCAGCAAG
TGATGGTGGT
TATCCCACTA
AACTGGCTTC
CGTAGTTAGG
G CT CTG CTAA TCTTAC CGGG CGGGC TGAAC
TACACCGAAC
TCCCGAAGGG
CAGGAGAGCG
AGTCCTGTCG
CTCGTCAGGG
ATGCGCCGCG
CCAAGGGTTG
CAATTCTTGG
AGGTCGAGGT
ACAAGGTATA
CGCCGAGGCG
TTAGGC TGGT TCATCTAC CT
GCCGGAAGCG
CGAACC'-CAG
C TAA CTTA CA AC CTGTCGTG GGTTTGC GTA
GCAACAGCTG
CGGTCCACGC
TAAC GGC GGG
CCGAGATATC
AG CAGAGC GC C CAC CAC TT C TC CTGTTAC C
TTGGACTCAA
GGGGGGTTCG
TGAGATAC CT
AGAAAGGCGG
CACGAGGGAG
GGTTT CGC CA
GGGCGGAGCC
TGCGGCTGCT
GTTTGCGCAT
AGTGGTGAAT
GGCCCGGCTC
GGGCGGCGCC
GCATAAATCG
AAGAGCCGCG
GC CTGGACAG
AGAAGAATCA
C kAGAC GTAG
TTAATTGCGT
C CAGCTGCAT
TTGGGCGCCA
ATTGCCCTTC
TGGTTTGCCC
ATATAACATG
CGCACCAACG
5301 5351 CAGCAGGCGA- AAATCCTGT'' AGCTGTCTTC GGTATCGTCG 5401 CGCAGCCCGG ACTCGGTAAT GGCGCGCATT GCGCCCAGCG CCATCTGATC 19 5451 5501.
5551 5601 5651 5701 5751 5801 5851 5901 SSS~ 5951 6001 6051 6101 6151 eee. 6201 6251 6301 6351 6401
GTTGGCAACC
TGGTTTGTTG
ATC GG CTGAA
CAGACGCGCC
GGTGACCCAA
TGGGAGAAAA
TAACGCCGGA
CATCCAGCGG
TTGTGCACCG
CACCACCACG
CAATTTGC GA AG CAA CGAC T
ATTCAGCTCC
CGTGGCTGGC
G CATACTCTG
GAATTGACTC
ACCATTCGTA
TGCAAGCTGA
GTCAGGCAGC
TGCATAATTC
AGCATCGCAG
AAAACCGGAC
TTTGATTGCG
GAGACAGAAC
TGCGACCAGA
TAATACTGTT
ACATTAGTGC
ATAGTTAATG
C CGCTTTACA CTGGCAC CCA
CGGCGCGTGC
GTTTGCCCGC
GCCATCGCCG
CTGGTTCACC
CGACATCGTA
TCTTCCGGGC
TGGTGTCAAC
TCGGAGCTTA
CATCGGAAGC
GTGTCGCTCA
TGGGAACGAT
ATGGCACTCC
AGTGAGATAT
TTAATGGGC C
TGCTCCACGC
GATGGGTGT C
AGGCAGCTTC
ATCAGCCCAC
GGCTTCGACG
GTTGATCGGC
AGGGCCAGAC
CAGTTGTTGT
CTTCCACTTT
ACGCGGGAAA
TAAC GTTACT
GCTATCATGC
GTAAATGCCC
TC GACTGCAC
TGTGGTATGG
AGGCGCACTC
GCCCTCATTC
AGTCGCCTTC
TTATGCCAGC
CGCTAACAGC
CCAGTCGCGT
TGGTCAGAGA
CACAG CAATG TGAC GC GTTG
CCGCTTCGTT
GCGAGATTTA
TGGAGGTGGC
GCCACGCGGT
TTCCCGCGTT
CGGTCTGATA
GGTTTCACAT
CATACCGCGA
GTTGCCCTTC
GGTGCAC CAA
CTGTGCAGGT
C CGTTCTGGA
AGCATTTGCA
CCGTTCCGCT
CAGCCAGACG
GCGATTTGCT
ACCGTCTTCA
CAT CAAGAAA
GCATCCTGGT
CGCGAGAAGA
CTACCATCGA
ATCGCCGCGA
AACGCCAATC
TGGGAATGTA
TTCGCAGAAA
AGAGACAC CG TCAC CAC CCT
AAGGTTTTGC
GCGCGCGAAT
TGCTTCTGGC
CGTAAATCAC
TAATGTTTTT
S
S. S S S S 5 55 6451 TGCGCCGACA TCATAACGGT TCTGGCA
Claims (10)
1. A fusion protein in which one partner contains the outer membrane protein A (OmpA) of Gram-negative bacteria and the other partner contains proteins larger than amino acids (AA).
2. A fusion protein as claimed in claim 1, wherein the OmpA derives from E.coli or S.dysenteriae or is composed of a hybrid of these OmpA. S. S
3. A fusion protein wherein a protein larger than 20 AA is inserted into one of the four outer apical regions of the OmpA of Gram-negative bacteria.
4. A fusion protein as claimed in claim 3, wherein the OmpA derives from E.coli or S.dysenteriae or is composed of a hybrid of these OmpA. go". A fusion protein as claimed in claim 3, wherein e•. additionally a stalk corresponding to the three-dimen- sional folding domain from the amino- or carboxy-terminal region of the influenza hemagglutinin (about AA 1-50 and AA 280-510) is inserted in front of the amino- or behind the carboxy-terminal end of the inserted protein.
6. A fusion protein as claimed in claim 3, wherein additionally a sequence which can be cleaved by protease is placed in front of the amino- and behind the carboxy- terminal end of the protein insert.
7. A fusion protein as claimed in claim 6, wherein a sequence which can be cleaved by collagenase is inserted.
8. Gram-negative bacteria which are transformed with vectors which code for fusion proteins as claimed in any one of claims 1 to 7.
9. A process for the preparation of fusion proteins as I I I 21 I claimed in any one of claims 1 to 7, which comprises the DNA sequences coding for the peptides or proteins invol- ved being attached together in the correct reading frame, ligated into an expression vector, Gram-negative bacteria being transformed therewith and expression being brought about in the transformants. The process as claimed in claim 9, wherein cells of E.coli, S.typhi or S.typhimurium are transformed with a hybrid plasmid for the expression of the fusion proteins as claimed in claim 1, 2, 3, 4, 5, 6 or 7.
11. The use of the fusion proteins as claimed in claim 1, 2, 3, 4, 5, 6 or 7 as vaccine.
12. The use of the transformed Gram-negative bacteria as claimed in claim 8, which have been induced with an inducer, as oral live vaccine. o* S OD 0 So S.o. 'SOS ad S S 55 5 a is DATED this 22nd day of August 1989. BEHRINGWERKE AKTIENGESELLSCHAFT 0O S S OS 55 S ~a S S S I *5 WATERMARK PATENT TRADEMARK AITORNEYS 50 QUEEN STREET MELBOURNE. VIC. 3000.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE3828666A DE3828666A1 (en) | 1988-08-24 | 1988-08-24 | EXPRESSION OF FUNCTIONAL HOMOLOGERS AND HETEROLOGICAL PROTEINS ON THE OUTSIDE MEMBRANE OF E.COLI AND OTHER GRAM-NEGATIVE BACTERIA |
| DE3828666 | 1988-08-24 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| AU4016089A AU4016089A (en) | 1990-03-01 |
| AU614174B2 true AU614174B2 (en) | 1991-08-22 |
Family
ID=6361472
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU40160/89A Ceased AU614174B2 (en) | 1988-08-24 | 1989-08-23 | The expression of homologous and heterologous proteins, which are able to function, on the outer membrane of e. coli and other gram-negative bacteria |
Country Status (8)
| Country | Link |
|---|---|
| EP (1) | EP0355737A3 (en) |
| JP (1) | JPH02135095A (en) |
| KR (1) | KR900003372A (en) |
| AU (1) | AU614174B2 (en) |
| DE (1) | DE3828666A1 (en) |
| DK (1) | DK416489A (en) |
| FI (1) | FI893932A (en) |
| PT (1) | PT91520A (en) |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5821088A (en) * | 1990-05-11 | 1998-10-13 | Siga Pharmaceuticals, Inc. | Use of gram-positive bacteria to express recombinant proteins |
| EP0474891A1 (en) * | 1990-09-08 | 1992-03-18 | BEHRINGWERKE Aktiengesellschaft | Vectors for expression of malarial antigens on the surface of Salmonella vaccine strains |
| CA2123675A1 (en) * | 1991-11-15 | 1993-05-27 | David W. Niesel | Membrane expression of heterologous genes |
| US5356797A (en) * | 1991-11-15 | 1994-10-18 | Board Of Regents, The University Of Texas | Membrane expression of heterologous genes |
| WO1993024636A1 (en) * | 1992-05-29 | 1993-12-09 | The University Of British Columbia | Use of protein oprf for bacterial cell surface expression of oligopeptides |
| FR2718452B1 (en) | 1994-04-06 | 1996-06-28 | Pf Medicament | Element of immunogen, immunogenic agent, pharmaceutical composition and method of preparation. |
| DE69637659D1 (en) * | 1995-05-15 | 2008-10-09 | Abbott Lab | ANCHORED EXPRESSION OF HETEROLOGIC PROTEINS |
| GB9718591D0 (en) * | 1997-09-03 | 1997-11-05 | Zeneca Ltd | Methods |
| NO20010238D0 (en) * | 2001-01-12 | 2001-01-12 | Unifob Stiftelsen Universitets | Systems for presentation on the surface of methanotrophic bacteria |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IL60184A (en) * | 1979-05-31 | 1984-05-31 | Schering Ag | Process for the specific cleavage of protein sequences from proteins |
-
1988
- 1988-08-24 DE DE3828666A patent/DE3828666A1/en not_active Withdrawn
-
1989
- 1989-08-18 EP EP19890115263 patent/EP0355737A3/en not_active Withdrawn
- 1989-08-22 FI FI893932A patent/FI893932A/en not_active Application Discontinuation
- 1989-08-23 PT PT91520A patent/PT91520A/en unknown
- 1989-08-23 AU AU40160/89A patent/AU614174B2/en not_active Ceased
- 1989-08-23 DK DK416489A patent/DK416489A/en not_active Application Discontinuation
- 1989-08-24 KR KR1019890012043A patent/KR900003372A/en not_active Application Discontinuation
- 1989-08-24 JP JP1218439A patent/JPH02135095A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| PT91520A (en) | 1990-03-08 |
| KR900003372A (en) | 1990-03-26 |
| JPH02135095A (en) | 1990-05-23 |
| EP0355737A3 (en) | 1991-10-30 |
| EP0355737A2 (en) | 1990-02-28 |
| FI893932A (en) | 1990-02-25 |
| DK416489D0 (en) | 1989-08-23 |
| DE3828666A1 (en) | 1990-03-01 |
| AU4016089A (en) | 1990-03-01 |
| FI893932A0 (en) | 1989-08-22 |
| DK416489A (en) | 1990-02-25 |
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