EP0951539A1 - Systeme vectoriel de clonage d'adn - Google Patents

Systeme vectoriel de clonage d'adn

Info

Publication number
EP0951539A1
EP0951539A1 EP97953645A EP97953645A EP0951539A1 EP 0951539 A1 EP0951539 A1 EP 0951539A1 EP 97953645 A EP97953645 A EP 97953645A EP 97953645 A EP97953645 A EP 97953645A EP 0951539 A1 EP0951539 A1 EP 0951539A1
Authority
EP
European Patent Office
Prior art keywords
sequence
protein
cloning
restriction
vector system
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Withdrawn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
EP97953645A
Other languages
German (de)
English (en)
Inventor
Wolf M. Prof. Dr. Bertling
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.)
Individual
Original Assignee
Individual
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 Individual filed Critical Individual
Publication of EP0951539A1 publication Critical patent/EP0951539A1/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Withdrawn legal-status Critical Current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
    • 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
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

Definitions

  • the invention relates to a vector system for cloning.
  • Common vectors used for cloning in prokaryotic systems usually contain the following features: a selection gene, e.g. the gene coding for ampicillin resistance, a marker gene which e.g. due to the color reaction, as with the lacZ gene, allows a differentiation between vectors with and without insert, and above all an origin of repii ca ti on.
  • a selection gene e.g. the gene coding for ampicillin resistance
  • a marker gene which e.g. due to the color reaction, as with the lacZ gene, allows a differentiation between vectors with and without insert, and above all an origin of repii ca ti on.
  • the vector system according to lit. c consists of a sequence of nucleotides which codes for the expression of a fusion protein.
  • the foreign protein sequence is here directly linked to the sequence for an enzyme and is not separated by other structures during the cloning process.
  • these residues are followed by a recognition sequence for a proteinase, for example the factor Xa.
  • a proteinase for example the factor Xa.
  • these proteinase interfaces enable the fusion protein to be split into the affinity portion and the foreign protein which is actually to be expressed.
  • this proteinase interface usually follows a multiple cloning sequence, into which the sequence to be expressed is cloned, further, often undesirable amino acids remain on the foreign protein to be expressed after the fusion protein has been processed.
  • the object of the invention is to provide a new vector system for cloning foreign proteins, which avoids the disadvantages of the prior art. This object is solved by the features of claims 1 and 10. Advantageous configurations result from the features of claims 2 - 9 and 11.
  • the use of the cloning vector system presented here now makes it possible, in a subsequent digestion and religation step, to use the foreign protein sequence to be expressed directly with an endoproteinase recognition sequence. This ensures that the foreign protein sequence to be expressed is released from an expressed fusion protein after digestion with the enzyme recognizing the corresponding protein sequence without further portions.
  • the recognition sequence for the enzyme BcgI was used to clone it within the multiplicity clone, in which the foreign sequence can later be inserted with any enzyme.
  • BcgI is one of the enzymes, so far the only commercially available enzyme that cut (10/12 nucleotides) from their recognition sequence both before and after this sequence.
  • a subsequent religation thus generates sequences from which 34 base pairs are removed.
  • targeted cloning e.g. the last nucleotide of the endoproteinase recognition sequence and the first nucleotide of the first codon of the protein to be cloned immediately together.
  • Example sequences are listed in Example 1 when listing some applications.
  • BcgI system has its tendency to lead to small deletions (usually 3 nucleotides) at the interface, depending on the buffer concentration, in 10-50% of cases. This means that in these cases the first amino acid of the foreign protein to be expressed, usually methionine, after digestion with the enzyme recognizing the corresponding protein sequence is no longer part of the foreign protein to be expressed, which is released in the process.
  • Fig. 4 shows a fourth sequence using the example of Clal,.
  • FIG. 7 shows a sixth sequence which contains a Bcgl recognition site.
  • the first sequence shown in Fig. 1 allows cloning in all three reading frames.
  • this enzyme can be used, the fragment filled up to a blunt end and cloned onto the starting construct cut and also filled up with AccI. This is shown in FIG. 3 using the example of BamHI.
  • the first nucleotide of the sequence to be expressed immediately follows the Xa protease recognition site.
  • Example 1 Design of the vector system.
  • Example 2 Cloning of a foreign protein, here the structural protein VP1 of the polyomavirus, into this cloning vector system.
  • VP1 contains an interface suitable for cloning (BamHI) at a distance of 6 nucleotides before its methionine start codon. Another interface necessary for cloning, SphI, follows its coding sequence at a distance of 59 nucleotides.
  • the cloning vector system as well as the construct comprising the VPl coding region were cut with the appropriate restriction endonucleases, Ac-cl / blunt and SphI.
  • the coding sequence was ligated into the cloning vector system thus prepared, transformed and amplified by growth in E. coli cells (XL1 blue (laclq), preventing expression in the absence of IPTG). Larger quantities of the plasmid construct produced in this way were isolated and purified for further processing.
  • the purified construct was then digested with the enzyme BcgI, separated in an agarose gel and purified and then religated and again transformed and amplified in bacteria which are particularly suitable for expression (RB791, a derivative of W3110 with ladqL ⁇ ).
  • RB791 a derivative of W3110 with ladqL ⁇ .
  • Example 3 Expression of a foreign protein
  • VP1 fusion protein Constructs encoding a VP1 fusion protein were transformed into bacterial cells (RB79 1) and grown in an overnight culture. Bacterial cultures up to a density of approx. 0.8A 60 o were used from this overnight culture. Expression of the fusion protein comprising the histidine residues, the factor Xa recognition site and VP1, was then induced by adding IPTG. After 6 hours of induction, total protein was harvested and an aliquot applied to gel to check induction efficiency. The main part of this protein mixture was bound to a nickel chelate column according to the manufacturer's instructions (Qiagen) and washed there with various buffers. The fusion protein can be eluted from the column using a solution which contains 50 mM ETGA. In our case, however, the pure expression protein VPl was released by subsequent digestion with the addition of endoproteinase factor Xa.

Landscapes

  • Genetics & Genomics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne un système vectoriel de clonage comprenant une séquence de nucléotides qui contient une séquence de fusion et un ou plusieurs sites de reconnaissance d'endonucléase de restriction pour des endonucléases de restriction qui provoquent une coupure en dehors de leurs sites de reconnaissance, en plus d'un ou de plusieurs autres sites de reconnaissance s'utilisant pour le clonage d'une protéine exogène, ainsi que la séquence requise pour la protéine exogène voulue. La séquence de protéine exogène se trouve directement sur la séquence de fusion, suite à une restriction avec les endonucléases de restriction, puis à une religation.
EP97953645A 1996-12-20 1997-12-18 Systeme vectoriel de clonage d'adn Withdrawn EP0951539A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE19653498A DE19653498C1 (de) 1996-12-20 1996-12-20 Klonierungsvektor-System mit Fusionssequenz
DE19653498 1996-12-20
PCT/DE1997/002963 WO1998028415A1 (fr) 1996-12-20 1997-12-18 Systeme vectoriel de clonage d'adn

Publications (1)

Publication Number Publication Date
EP0951539A1 true EP0951539A1 (fr) 1999-10-27

Family

ID=7815668

Family Applications (1)

Application Number Title Priority Date Filing Date
EP97953645A Withdrawn EP0951539A1 (fr) 1996-12-20 1997-12-18 Systeme vectoriel de clonage d'adn

Country Status (4)

Country Link
US (1) US6187589B1 (fr)
EP (1) EP0951539A1 (fr)
DE (1) DE19653498C1 (fr)
WO (1) WO1998028415A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8293503B2 (en) 2003-10-03 2012-10-23 Promega Corporation Vectors for directional cloning

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6906173B2 (en) 1998-11-03 2005-06-14 Migenix Corp. Production of adenine nucleotide translocator (ANT), novel ANT ligands and screening assays therefor
EP1049780A1 (fr) 1998-11-03 2000-11-08 Mitokor Production d'un translocateur de nucleotide d'adenine (ant), nouveaux ligands a l'ant et essais de criblage associes
EP1783221A1 (fr) * 2000-05-11 2007-05-09 MIGENIX Corp. Production d'un translocateur de nucléotide d'adenine (ANT), nouveaux ligands à l'ANT et essais de DE criblage associés
DE10145553B4 (de) * 2001-09-16 2006-06-08 Gene Architects Ag Nukleinsäure für einen Klonierungsvektor
CA2541177A1 (fr) * 2003-10-03 2005-09-22 Promega Corporation Vecteurs pour le clonage directionnel

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB8412517D0 (en) * 1984-05-16 1984-06-20 Nagai K Recombinant fusion proteins
NZ224663A (en) * 1987-05-28 1990-11-27 Amrad Corp Ltd Fusion proteins containing glutathione-s-transferase and expression of foreign polypeptides using glutathione-s-transferase encoding vectors
GB2212160B (en) * 1987-11-13 1991-12-11 British Bio Technology Dna sequences encoding the bspmi recognition site.
US5196524A (en) * 1989-01-06 1993-03-23 Eli Lilly And Company Fusion reporter gene for bacterial luciferase
US5200336A (en) * 1990-07-02 1993-04-06 New England Biolabs, Inc. Restriction endonuclease obtainable foam bacillus coagulans and a process for producing the same
JP3277523B2 (ja) * 1991-09-13 2002-04-22 株式会社日立製作所 因子ＸａリンカーＤＮＡ
US5434063A (en) * 1991-12-20 1995-07-18 The United States Of America As Represented By The United States Department Of Energy Sequential cloning of chromosomes

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9828415A1 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8293503B2 (en) 2003-10-03 2012-10-23 Promega Corporation Vectors for directional cloning
US8367403B2 (en) 2003-10-03 2013-02-05 Promega Corporation Vectors for directional cloning
US9018014B2 (en) 2003-10-03 2015-04-28 Promega Corporation Vectors for directional cloning
US9371531B2 (en) 2003-10-03 2016-06-21 Promega Corporation Vectors for directional cloning
US9469857B2 (en) 2003-10-03 2016-10-18 Promega Corporation Vectors for directional cloning

Also Published As

Publication number Publication date
US6187589B1 (en) 2001-02-13
WO1998028415A1 (fr) 1998-07-02
DE19653498C1 (de) 1998-07-23

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