EP0424429A1 - Nouveaux vecteurs d'expression - Google Patents

Nouveaux vecteurs d'expression

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Publication number
EP0424429A1
EP0424429A1 EP89907811A EP89907811A EP0424429A1 EP 0424429 A1 EP0424429 A1 EP 0424429A1 EP 89907811 A EP89907811 A EP 89907811A EP 89907811 A EP89907811 A EP 89907811A EP 0424429 A1 EP0424429 A1 EP 0424429A1
Authority
EP
European Patent Office
Prior art keywords
amino acids
plasmid
gene
sequence coding
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP89907811A
Other languages
German (de)
English (en)
Inventor
Tamás LUKACSOVICH
Pál VENETIANER
András OROSZ
Gabriella Baliko
Imre Boros
Eva Balla
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Richter Gedeon Vegyeszeti Gyar Nyrt
Original Assignee
Richter Gedeon Vegyeszeti Gyar RT
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Richter Gedeon Vegyeszeti Gyar RT filed Critical Richter Gedeon Vegyeszeti Gyar RT
Publication of EP0424429A1 publication Critical patent/EP0424429A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • 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
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
    • C12N9/2471—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01023—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones

Definitions

  • the present invention relates to novel expression vectors comprising besides the usual transcription and translation regulating sequences, i.e. promoter, operator, ribosomal binding site, translation start codon, transcription terminators, etc., a sequence coding for the part of ⁇ -galactosidase comprising
  • the invention also relates to the method for production of
  • the method of in vitro DNA recombination can be used for introducing genes of whatever origin and information content into bacterial cells.
  • the expression vectors can assure the foreign DNA to stably exist in the cell, and express the information encoded.
  • the enzymes of the bacterial cell synthesize mRNA from the DNA during transcription, followed by synthetizing protein therefrom
  • RNA polymerase recognizes this promoter and binds to it during the initiation of transcription.
  • the method of in vitro DNA recombination has a practical value only if the target protein is biosynthesized in sufficient amount, that is if both the transcription and translation has proper intensity and the produced mRNA and biosynthesized protein is properly stable in the host cell.
  • the firstzi can be overcome by a promoter regulatable and initiatable at a given moment which is turned on by the user when the bacterial cells reach proper density. At this stage it does not mean anylock if only the synthesis of the product is going further on in the cells, there is no more division of cells, the life conditions - just because of the synthesis and toxicity of the product - are not ideal for the host cell.
  • the mRNA and protein level protection is achieved by "masking" the gene in question in such a way that a part of a gene of bacterial origin is fused to the 5'-end of the gene of the product.
  • this protecting peptide is a part of the ⁇ -galactosidase enzyme, the relative amount of which is very high in the fusion product since a sequence of 150 amino acids is needed for protection, as a minimum.
  • This fact represents a serious disadvantage in the purification of the product since the relative amount of the useful protein is small in the fusion protein and the most widely used separation procedure, the BrCN cleavage, results in many fragments, from which the selection of the authentic one is difficult.
  • the mRNA level protection can be achieved if the region coding for the monoton amino acids is inserted after a short ⁇ -galactosidase DNA sequence (coding for 15 - 20 amino acids) and before the gene to be expressed.
  • This "sandwich" arrangement assures the sufficient mRNA and protein level protection.
  • Our conclusion is that the ⁇ -galactosidase coding part is important for the mRNA level protection while the homopolymer amino acid tail has greater importance on the protein level.
  • coding for a homopolymer of amino acids comprising introducing a sequence coding for 15-20 amino acids of ⁇ -glactosidase, the repetition of the operator sequence or any part thereof, a further ribosomal binding site, a translation start codon, a sequence coding for a homopolymer oligopeptide comprising
  • the vector carries a very strong regulatable promoter
  • homopolymer amino acid tail syntesized by the sequence coding for the homopolymer of amino acids, both located before the gene to be expressed;
  • the length of the tail is only about 40 amino acids that makes the purification of the product significantly easier;
  • the undesired fused protein parts can be removed from the product protein by a simple BrCN cleavage
  • the vector can be used for the expression of any other protein gene containing ClaI linker or any protein gene inserted into the ClaI site of the vector (other restriction sites of the linker can also be used of course);
  • polypeptide very high level/expression could be effected by the vector (25-30 % of the total protein content of the cell).
  • the method according to the present invention elaborated for the construction of the above mentioned expression vector suitably comprises the following steps, detailed later.
  • a peptide is selected, the gene coding for which is available in a cloned form and which is known to be very instable in E. coli cell (for example the human proinsulin).
  • the gene starting with the Cla I linker is cloned into a suitable plasmid following the lac regulator sequences (operator and ribosomal binding site).
  • plasmid a synthetic oligonucl ⁇ otid is inserted which contains the sequencial coding region of some amino acids threonin.
  • the triplets of the threonins are in reading frame with the gene and, on the other hand, the Cla I site regenerates only at the end of the oligonucleotide being closer to the gene.
  • the plasmid is linearized, then deletions are made with Bal 31 exonuclease in such circumstances that the enzyme should digest only some 10 nucleotides, then the plasmid is recircularized with DNA ligase.
  • the produced fusion peptid involves the protein to be expressed.
  • pSZI 153 pERVI/23 PLH4, pERVI /23/+ATG/.
  • the plasmid named pSZI 153 is described in our Hungarian patent application No. 4363/84, the other two ones are the members of the vector family described in our Hungarian patent application No. 4111/87.
  • the DNA region coding for human proinsulin is cut out from plasmid pSZI 153 with ClaI and HindIII restriction enzymes, where on the removed DNA fragment, the nucleotide triplet originating from the ClaI linker is directly followed by the codon TTT coding for the first amino acid of the protein.
  • the DNA fragment (proinsulin gene) produced by digestion with ClaI, HindIII restriction enzymes is cloned into the ClaI-HindIII site of the polylinker region of plasmid pER23(+ATG).
  • a synthetic double stranded oligonucleotide is inserted that contains sequentially the codons of seven threonin amino acids and carries two sticky ends that can be ligated to a plasmid digested with ClaI enzyme.
  • the oligonucleotide is designed in such a way that, in case of insertion in a correct orientation, the threonin codons are in reading frame with the proinsulin gene and the
  • ClaI restriction site should be regenerated only on the end of the oligonucleotide closer to the gene.
  • plasmid is recircularised with DNA ligase in the presence of large amount of EcoRI linker phosphorylated by
  • the Intermediate V plasmid is digested with EcoRI restriction enzyme and series of deletions are generated with Bal31 exonuclease in both directions from the EcoRI site.
  • the member of the series is selected, in which the Bal 31 enzyme digested some dozens of nucleotides in both directions.
  • the distance between the ⁇ -peptide coding region and the seven threonine codons is reduced.
  • the resulting mixture of plasmids is transformed into E. coli IM 107 cells and clones are selected expressing foreing protein belonging to the desired size-range in large amount.
  • a suitable clone was selected and the end-points of the deletion was determined by DNA sequencing on the plasmid prepared therefrom.
  • the concrete construction and sequence are shown on fig. 2/a, Fig. 2/b and Fig. 3.
  • the produced fusion protein is strongly reacting with antibodies specific for the human proinsulin protein.
  • DNA polymerase I Large Fragment were from New England Biolabs, bacterial alkaline phosphatase (BAP) was from Worthington, the pancrease RN-ase and the lysozyme were from Reanal.
  • T 4 induced p ⁇ lynucleotide ligase was prepared by the method of Murray et al. (Murray, N.E., Bruce S.A. and Murray, K.: Molecular cloning of the DNA ligase gene from bacteriophage T 4 . D. Mol. Biol.,
  • E. coli K12 JM107 (Yanisch-Perron, C. et al.: Improved M13 phage cloning vectors and host strains nucleotide sequences of the M14 mp 18 and pUC19 vectors. Gene, 33 , 103-119 (1985)).
  • Escherichia coli strains were grown in liquid media completed with 10 g Bacto-tryptone, 5 g Bacto Yeast extract and 5 g NaCl per litre. Solid culture medium was prepared by adding 15 g Bacto-agar per litre to the above liquid medium.
  • Indicator plates suitable for the estimation of the expression of the N-terminal part of the ⁇ -galactosidase enzyme ( ⁇ -peptide) were composed of the following components per litre:
  • the cells containing the plasmid carrying the ⁇ -lactamase gene were grown in a medium containing 100 ⁇ g/ml ampicillin.
  • Isolation of plasmid DNA was made by growing the bacterium strain carrying the plasmid in question in a medium containing 100 ⁇ g/ml ampicillin and, at the time of reaching 0.7-0.8 OD 600nm' adding 170 ⁇ g/ml chloramphenicol to the culture medium for the amplification of the plasmid.
  • clear lysate was prepared by the method described by Clewell and Helinski (Clewell, D.B. and Helinski, D.R., (1969)) Supercoiled circular DNA-protein complex in Escherichia coli:
  • reaction mixture (30-40 ⁇ l) comprising 0.5-1.0 ⁇ g DNA, 66 mM Tris-HCl (pH 7.6), 5 mM MgCl 2 , 5 mM dithiothreitol, 1 mM ATP and 1 unit induced T 4 polynucleotid ligase was used.
  • the ligation was carried out at 14 oC for 2-3 hours /Maniatis T., Fritsch, E.F., Sambrook, J . (1982) Molecular Cloning, Cold
  • Joning of fragments with blunt ends was carried out in a buffer containing 30-40 ⁇ g/ml DNA, 25 mM Tris-HCl (pH 7.4), 5 mM MgCl 2 , 5 mM dithiothreitol, 0.25 mM spermidin, 1 mM ATP, 10 ⁇ g/ml BSA (Sigma, Type V). 4-6 units of induced T 4 polynucleotide kinase were given to the reaction mixture and it was incubated at 14 oC for 8 - 12 hours.
  • the DNA fragments were digested with BAL31 nuclease in a final concentration of 100 ⁇ g/ ⁇ l in a reaction mixture containing 600 mM NaCl, 12 mM CaCl 2 , 20 mM Tris-Hcl (pH 8.0), 1.0 mM EDTA at 30 oC. Depending on the desired extent of the shortening, 0.4 - 1.2 units of enzyme were added to 1.0 ⁇ g of DNA. In each case test preparation was made with the given
  • DNA fragments and end-labeling with polynucleotide kinase using ⁇ - 32 P-ATP as well as the determination of the nucleotid sequence was performed according to the protocol described by Maxam A. and Gilbert, w. /Sequencing end-labelled DNA with basespecific chemical cleavages. Meth. Enzymol. 65, pp. 499 - 560 (1980)/.
  • the fusion protein containing insulin was demonstrated by the immunoblot method /Towbin et al.: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. Procedure and some applications, Proc. Natl. Acad. Sci. USA, 76, 4350 (1979)/.
  • Ap R beta-lactamase gene, providing ampicillin resistance

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
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  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Plant Pathology (AREA)
  • Endocrinology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Diabetes (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

L'invention concerne de nouveaux vecteurs d'expression formés à partir d'un dérivé de plasmide pERVI/23 comprenant, après le promoteur, l'opérateur, le site de liaison ribosomique, et les régions de codon de départ de traduction du plasmide, et avant le gène à exprimer, une séquence codant pour 15 à 20 acides aminés de beta-galactosidase, la répétition de la séquence opérateur ou de n'importe quelle partie de celle-ci, un autre site de liaison ribosomique, un codon de départ de traduction, une séquence codant pour un homopolymère de 5 à 10 acides aminés, un autre codon ATG étant contenus dans ledit dérivé de plasmide. L'invention concerne également un procédé de construction des vecteurs d'expression précités. Les nouveaux vecteurs d'expression de l'invention permettent une production de protéines très efficace, sont en outre régulables de manière fiable, et, s'il y a un gène à exprimer, fournissent un ARN messager adapté et une stabilité de niveau protéique.
EP89907811A 1988-07-14 1989-07-11 Nouveaux vecteurs d'expression Withdrawn EP0424429A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
HU368588 1988-07-14
HU883685A HU200486B (en) 1988-07-14 1988-07-14 Process for producing expression vector

Publications (1)

Publication Number Publication Date
EP0424429A1 true EP0424429A1 (fr) 1991-05-02

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Application Number Title Priority Date Filing Date
EP89907811A Withdrawn EP0424429A1 (fr) 1988-07-14 1989-07-11 Nouveaux vecteurs d'expression

Country Status (6)

Country Link
EP (1) EP0424429A1 (fr)
JP (1) JPH04500453A (fr)
KR (1) KR920007684B1 (fr)
AU (1) AU3961989A (fr)
HU (1) HU200486B (fr)
WO (1) WO1990000610A1 (fr)

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
HU197595B (en) * 1987-09-16 1989-04-28 Richter Gedeon Vegyeszet Process for producing expression vectors

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9000610A1 *

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Publication number Publication date
KR920007684B1 (ko) 1992-09-14
AU3961989A (en) 1990-02-05
KR900702038A (ko) 1990-12-05
JPH04500453A (ja) 1992-01-30
HU200486B (en) 1990-06-28
WO1990000610A1 (fr) 1990-01-25

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