AU624667B2 - Plasmids with a translation start-stop-start codon configuration for the expression of proteins in e. coli - Google Patents
Plasmids with a translation start-stop-start codon configuration for the expression of proteins in e. coli Download PDFInfo
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- AU624667B2 AU624667B2 AU47202/89A AU4720289A AU624667B2 AU 624667 B2 AU624667 B2 AU 624667B2 AU 47202/89 A AU47202/89 A AU 47202/89A AU 4720289 A AU4720289 A AU 4720289A AU 624667 B2 AU624667 B2 AU 624667B2
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- 108090000623 proteins and genes Proteins 0.000 title claims description 25
- 241000588724 Escherichia coli Species 0.000 title claims description 19
- 102000004169 proteins and genes Human genes 0.000 title claims description 16
- 108020004705 Codon Proteins 0.000 title claims description 14
- 239000013612 plasmid Substances 0.000 title claims description 12
- 108091081024 Start codon Proteins 0.000 claims description 8
- 239000013613 expression plasmid Substances 0.000 claims description 8
- 108020004414 DNA Proteins 0.000 claims description 5
- 238000000034 method Methods 0.000 claims description 5
- 238000011144 upstream manufacturing Methods 0.000 claims description 4
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 238000010353 genetic engineering Methods 0.000 claims description 2
- 238000002255 vaccination Methods 0.000 claims description 2
- 229960005486 vaccine Drugs 0.000 claims description 2
- 108010052418 (N-(2-((4-((2-((4-(9-acridinylamino)phenyl)amino)-2-oxoethyl)amino)-4-oxobutyl)amino)-1-(1H-imidazol-4-ylmethyl)-1-oxoethyl)-6-(((-2-aminoethyl)amino)methyl)-2-pyridinecarboxamidato) iron(1+) Proteins 0.000 claims 1
- 235000009917 Crataegus X brevipes Nutrition 0.000 claims 1
- 235000013204 Crataegus X haemacarpa Nutrition 0.000 claims 1
- 235000009685 Crataegus X maligna Nutrition 0.000 claims 1
- 235000009444 Crataegus X rubrocarnea Nutrition 0.000 claims 1
- 235000009486 Crataegus bullatus Nutrition 0.000 claims 1
- 235000017181 Crataegus chrysocarpa Nutrition 0.000 claims 1
- 235000009682 Crataegus limnophila Nutrition 0.000 claims 1
- 235000004423 Crataegus monogyna Nutrition 0.000 claims 1
- 240000000171 Crataegus monogyna Species 0.000 claims 1
- 235000002313 Crataegus paludosa Nutrition 0.000 claims 1
- 235000009840 Crataegus x incaedua Nutrition 0.000 claims 1
- 238000009007 Diagnostic Kit Methods 0.000 claims 1
- 239000002299 complementary DNA Substances 0.000 claims 1
- 210000002837 heart atrium Anatomy 0.000 claims 1
- 244000144972 livestock Species 0.000 claims 1
- 108020004999 messenger RNA Proteins 0.000 description 5
- 150000001413 amino acids Chemical class 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 239000013598 vector Substances 0.000 description 4
- 101710146739 Enterotoxin Proteins 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 239000000147 enterotoxin Substances 0.000 description 3
- 231100000655 enterotoxin Toxicity 0.000 description 3
- 239000013604 expression vector Substances 0.000 description 3
- 229920001184 polypeptide Polymers 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- 102000004196 processed proteins & peptides Human genes 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 239000000284 extract Substances 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 230000002163 immunogen Effects 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 210000000936 intestine Anatomy 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 239000008174 sterile solution Substances 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 241000478345 Afer Species 0.000 description 1
- 102000009016 Cholera Toxin Human genes 0.000 description 1
- 108010049048 Cholera Toxin Proteins 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- 102000030621 adenylate cyclase Human genes 0.000 description 1
- 108060000200 adenylate cyclase Proteins 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 239000003782 beta lactam antibiotic agent Substances 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 108010055409 ganglioside receptor Proteins 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 101150109249 lacI gene Proteins 0.000 description 1
- MJIHNNLFOKEZEW-UHFFFAOYSA-N lansoprazole Chemical compound CC1=C(OCC(F)(F)F)C=CN=C1CS(=O)C1=NC2=CC=CC=C2N1 MJIHNNLFOKEZEW-UHFFFAOYSA-N 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000006919 modified lb Substances 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 230000006337 proteolytic cleavage Effects 0.000 description 1
- 230000000384 rearing effect Effects 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- C07K14/245—Escherichia (G)
-
- 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
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- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Description
Form COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952-69 COMPLETE SPECIFICATION
(ORIGINAL)
Class Ir Application Number: Lodged: tJ nt. Class Complete Specification Lodged: Accepted: Published: Priority Helated Art Name of Applicant: BEHRINGWERKE AKTIENGESELLSCHAFT Address of Applicant :D-3550 Marburg, Federal Republic of Germany Actual Inventor Address for Service MICHAEL BROKER WATERMARK PATENT TRADEMARK ATTORNEYS.
290 Burwood Road, Hawthorn, Victoria, Australia Complete Specification for the invention entitled: PLASMIDS WITH A TRANSLATION START-STOP-START CODON CONFIGURATION FOR THE EXPRESSION OF PROTEINS IN E. coli The following statement is a full description of this invention, including the best method of performing it known to 1 _I _1~1~ BEHRINGWERKE AKTIENGESELLSCHAFT 88/B 047 Ma 740 Dr. Lp/rd Plasmids with a translation start-stop-start codon configuration for the expression of proteins in E. coli The invention relates to expression plasmids which bring about a considerable increase in the expression of proteins, in particular of the thermolabile E. coli enterotoxin subunit B This entails a strong promoter being placed upstream of the DNA coding for LT-B in such a way that the so-called Shine-Dalgarno sequence GGGA of the LT-B gene is located six base-pairs (bp) in front of the LT-B translation start, with retention of the stop codon TGA of the LT-A sequence which precedes in the natural case.
Infections with enterotoxinogenic Escherichia coli coli) are often the cause of diarrhea in humans and animals. Besides the thermostable E. coli enterotoxin, significant economic problems in pig rearing are caused in particular by the thermolabile E. coli enterotoxin Newborn pigs often become infected with pathogenic E. coli strains within a few hours afer birth, which leads to a high mortality rate. Vaccination with LT toxoid might contribute to reducing the infection rate and thus the losses on large stock farms.
The properties of LT are similar to that of choleratoxin.
The A subunits of both toxins influence membrane permeability via stimulation of the membrane-bound adenylate cyclase of the epithelial cells of the intestine. The binding of LT takes places via the B subunit (LT-B) to the GM ganglioside receptor on the surface of the mucosa cells of the intestine. LT is translated by a polycistronic messenger RNA (mRNA) which has separate ribosomebinding sites (RBS) Shine-Dalgarno sequences (SD) for the A and B subunits, which is probably the cause of the difference in the translation initiation rate of about 2 five B subunits to one A subunit. The gene for LT-B has been sequenced by Dallas and Falkow (Nature 288, 499-501, (1980)). The LT-B gene codes for a protein of 124 amino acids, with 22 amino-terminal amino acids acting as signal sequences and being eliminated during transport of LT-B through the cell membrane. The molecular weight of processed LT-B is 11,780 D. Since LT-B is not pathogenic but is immunogenic, a process has subsequently been developed to be able to produce LT-B in large amounts in apathogenic E. coli with the aid of molecular biological methods.
The intention was that the synthesis of LT-B take place by strong promoters which can be regulated in an advantageous manner, of which the tac promoter is a representative (De Boer, H.A. et al., in: Rodriguez et al., Promoters, Structure and Function, Praeger, New York, 1982, 462-481). On the other hand, the intention was to utilize the RBS of the LT-B gene in order to ensure a high degree of translation of the transcribed mRNA in E. coli.
The genes of the LT-A and LT-B subunits overlap over a region of four nucleotides (see Fig. The proximal A of the sequence ATGA is the third base within the TTALeu codon for LT-A, and the last three bases TGA code for the translation stop of LT-A. On the other hand, the proximal A of the sequence ATGA is the first base of the ATGet codon which codes for initiation of pre-LT-B translation, although in a different reading frame.
We have found that the exproesien e-f proteins, in par ticular LT-B, is considerably increased when an ression vector which reflects the aboveme n ed situation is employed. Vectors of this have, after a strong promoter, an ATG start owed by 5 to 10 triplets which in turn cont an SD sequence; attached thereto is a tra ion stop codon TGA followed by 1 to 5 triplets up the ATC ctart of thepolypcptid of interet. It is
N'
L j-- We have found that the expression of proteins, in particular LT-B, is considerably increased when an expression vector which reflects the abovementioned situation is employed. Vectors of this type have, after a strong promoter, a start codon, preferably ATG followed by 5 to 10 triplets which in turn contain an SD sequence; attached thereto is a translation stop codon, preferably TGA which may be followed by 1 to 5 triplets up to the start preferably ATG of the polypeptide of interest. It is possible that the increase in expression which is entirely unexpected due to the inserted stop triplet, (preferably TGA) occurs due to this start-stop-restart arrangement in a small 1 0 space.
Accordingly, the invention relates to expression plasmids which, after a strong promoter, have a first start codon followed by 5 to 10 triplets coding for amino acids, there being a second contained SD sequence, a stop codon being attached and, in a short space of 1 to 5 further tr;plets another start codon being reached, which is followed by the DNA of the polypeptide of interest.
The start codon may be ATG.
-<A
3 po ssi- ±e e-I--nn-s-ea e WR-e---w4-necn Is 7 tirely unexpected due to the inserted TGA stop triplet occurs due to this start-stop-restart arrangemetin a small space.
Accordingly, the invention relate -o expression plasmids which, after a strong promp er, have a first ATG start codon followed by 5 ,t6 10 triplets coding for amino acids, there bei ga second contained SD sequence, a stop codon bein attached and, in a short space of 1 to furth triplets another ATG start codon being reached, ich is followed by the DNA of the polypeptide of intoroest.
The invention furthermore relates to the use of such plasmids for the expression of proteins, in particular LT-B in E. coli and the genetic engineering processes therefor.
Finally, the invention is described in the examples and the patent claims.
Examples Example 1: Suitable starting plasmids The starting point for the preparation of suitable expression vectors was the plasmid pOB3 (Br6ker, Biotechniques, 6, 734, 1988). This plasmid is replicable in E.
coli in high copy number. The ampicillin-resistance gene makes selection and fermentation in the presence of p-lactam antibiotics possible. An ATG translation triplet is located at the 3' end of the tac promoter and is followed by some restriction sites and at the further 3' end by the strong ribosomal transcription terminators TjT 2
T
4 Example 2: Construction of an LT-B expression vector The plasmid pOB3 was digested with EcoRI and HindIII, and the EcoRI-HindIII DNA fragment which codes for LT-B was ligated into the plasmid. The new plasmid pMBl91 (Fig. 2) thus codes for expression of LT-B, regulated by the tac promoter. In addition, there is created a situation in which, determined by the ATG codon and the following triplets, a pentapeptide of the structure Met-Arg-Asn- Ser-Gly is synthesized in E. coli. There follows, four base-pairs from the 3' end of the TGA stop codon, a new ATG translation start signal, which is now part of the pre-LT-B sequence.
Owing to the translation start repeated shortly after the translation stop, there is created a situation which is related to the translation of the natural bicistronic mRNA which codes for LT-A and LT-B. There is synthesised, encoded by pMBl91 of an mRNA on which, just like in the authentic gene transcript, the Shine-Dalgarno sequence GGGA is located six bp upstream of the translation start of LT-B. In the present example, the TGA stop codon was 0* additionally incorporated in the LT-B reading frame, which is not necessary per se.
Example 3: Expression of LT-B The new plasmid pMBl91 was transformed into E. coli D29A1. Transformed cells were cultured on LB medium, and the tac promoter was induced with isopropylthiogalactoside (IPTG). Protein extracts from E. coli D29A1 pMBl91 were fractionated on SDS polyacrylamide gels. In comparison with extracts derived form non-induced cultures, a new band with an apparent molecular weight of about 12,000 D was visualized by Coomassie brilliant blue staining. It is possible, by incubating the cells in a buffer of pH 8.0 with 10 mM EDTA, to bring about release of LT-B into the buffer. It is possible with the aid of this new expression system to prepare large amounts of 5 immunogenic LT-B in apathogenic E. coli strains as starting material for a vaccine. Regulated expression can take place in a strain which has the genotype lacI
Q
However, it is also possible to employ E. coli strains in which there is constitutive production of LT-B. The yield of LT-B can be additionally increased by using for the fermentation a modified LB medium in place of the customary LB medium. This medium is made up as follows: 100 ml of a sterile solution of 0.17 M KH 2
PO
4 and 0.72 M K 2
HPO
4 are added to a sterile solution which contains 12 g of Bacto tryptone, 24 g of Bacto yeast extract and 4 ml of glycerol made up to 900 ml with H 2
O.
In comparison with conventional expression systems, the use of pMB191 results in an increase in LT-B expression by a factor of 100. In the present case the protein of interest is processed by elimination of the signal sequence. In general, a sequence of this type will be placed upstream in order to obtain a "mature" protein without additional effort.
Legends to Fig. 1 and Fig. 2 Fig. 1: Section of the DNA sequence of the LT gene. Junction between the protein-encoding LT-A and LT-B region. SD: Shine-Dalgarno sequence of LT-B.
Fig. 2: Diagrammatic representation of the vector pMB191. Pta,: tac promoter. Amp ampicillin resistance. Tets: part of the gene which confers resistance to tetracycline.
TjT 2 transcription terminator.
LTSD: Shine-Dalgarno sequence of the LT-B subunit. The arrow indicates the proteolytic cleavage of pre-LT-B.
Claims (9)
1. An expression plasmid for the expression of proteins in E. coli having the consecutive elements a strong promoter, a first start codon, 5 to 10 triplets which contain at the 3' end the Shine-Dalgarno (SD) sequence AGGA or GGGA or another sequence functioning as SD, a stop codon, a second start codon at a distance of 1-10 triplets, which are located in front of the DNA coding for the protein of interest
2. An expression plasmid as claimed in claim 1, where a signal sequence is located upstream of the protein of interest.
3. An exprssion plasmid as claimed in claim 1, where the tac promoter with the attached sequence ACAGGAAACAGACCATGCGG AAT TCG GGA TGA ATT LaCsD LTsD is inserted in front of the ATG sequence of the protein to be expressed.
4. An expression plasmid as claimed in claim 1, 2 or 3, where the LT-B cDNA sequence is employed.
A process for the preparation, by genetic manipulation, of proteins, which comprises an expression plasmid as claimed in claim 1, 2 or 3 being transformed into E. coli and then being expressed therein.
6. 'A process for the preparation of LT-B, which comprises an expression plasmid as claimed in claim 4 being trans- formed into E. coli and then being expressed therein.
7. A vaccine which contains LT-B prepared as claimed in 1- wNT -7
8. The use of LT-B prepared as claimed in claim 6 for the vaccination of productive livestock.
9. A diagnostic kit which contains LT-B prepared as claimed in claim 6. DATED this 21st day of 1)ecember 1989. BEHRINGWERKE AK1ENGESELLSCHAFT WATERMARK PATENT TRAJ)EMR ATTOCRNEYS "THE ATRIUM" T 290 BIJRWOOD ROAD HAWTHORN. VIC. 3122.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE3843894A DE3843894A1 (en) | 1988-12-24 | 1988-12-24 | PLASMIDE WITH TRANSLATION-START-STOP-START CODON CONFIGURATION FOR EXPRESSION OF PROTEINS IN E.COLI |
| DE3843894 | 1988-12-24 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| AU4720289A AU4720289A (en) | 1990-06-28 |
| AU624667B2 true AU624667B2 (en) | 1992-06-18 |
Family
ID=6370260
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU47202/89A Ceased AU624667B2 (en) | 1988-12-24 | 1989-12-22 | Plasmids with a translation start-stop-start codon configuration for the expression of proteins in e. coli |
Country Status (9)
| Country | Link |
|---|---|
| EP (1) | EP0379710A1 (en) |
| JP (1) | JPH02231087A (en) |
| KR (1) | KR900009988A (en) |
| AU (1) | AU624667B2 (en) |
| CA (1) | CA2006534A1 (en) |
| DE (1) | DE3843894A1 (en) |
| DK (1) | DK661489A (en) |
| IE (1) | IE894177L (en) |
| PT (1) | PT92656A (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3901681A1 (en) * | 1989-01-21 | 1990-07-26 | Behringwerke Ag | SIGNAL PEPTIDE FOR THE SECRETION OF PEPTIDES IN ESCHERICHIA COLI, METHOD FOR ITS EFFECT AND ITS USE |
| JPH03187383A (en) * | 1989-09-04 | 1991-08-15 | Takeda Chem Ind Ltd | Manifestation plasmid and use thereof |
| US7202077B2 (en) | 2001-04-13 | 2007-04-10 | Zymogenetics, Inc. | Methods for enhancing the translation and expression of recombinant proteins |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA1200773A (en) * | 1980-02-29 | 1986-02-18 | William J. Rutter | Expression linkers |
| DOP1985004337A (en) * | 1984-03-06 | 1990-09-17 | Lilly Co Eli | VECTORS AND METHOD FOR GENETIC EXPRESSION |
| CA1340372C (en) * | 1984-07-09 | 1999-02-02 | John D. Clements | Production of e. coli lt-b enterotoxin subunit |
-
1988
- 1988-12-24 DE DE3843894A patent/DE3843894A1/en not_active Withdrawn
-
1989
- 1989-12-21 PT PT92656A patent/PT92656A/en not_active Application Discontinuation
- 1989-12-21 EP EP89123613A patent/EP0379710A1/en not_active Withdrawn
- 1989-12-21 IE IE894177A patent/IE894177L/en unknown
- 1989-12-22 AU AU47202/89A patent/AU624667B2/en not_active Ceased
- 1989-12-22 JP JP1331486A patent/JPH02231087A/en active Pending
- 1989-12-22 DK DK661489A patent/DK661489A/en unknown
- 1989-12-22 CA CA002006534A patent/CA2006534A1/en not_active Abandoned
- 1989-12-22 KR KR1019890019220A patent/KR900009988A/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| JPH02231087A (en) | 1990-09-13 |
| CA2006534A1 (en) | 1990-06-24 |
| IE894177L (en) | 1990-06-24 |
| DK661489D0 (en) | 1989-12-22 |
| DK661489A (en) | 1990-06-25 |
| DE3843894A1 (en) | 1990-06-28 |
| AU4720289A (en) | 1990-06-28 |
| KR900009988A (en) | 1990-07-06 |
| EP0379710A1 (en) | 1990-08-01 |
| PT92656A (en) | 1990-05-31 |
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