EP0951539A1 - Vectorial cloning system of dna - Google Patents

Vectorial cloning system of dna

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Withdrawn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)
French (fr)
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/en
Withdrawn legal-status Critical Current

Links

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.

Abstract

The invention relates to a vectorial cloning system consisting of a sequence of nucleotides, containing a fusion sequence and one or several restriction endonucleases, recognition sites for restriction endonucleases cutting outside their recognition sites, in addition to containing one or several other restriction endonuclease recognition sites which can be used to clone a foreign protein as well as the sequence for the desired foreign protein, wherein the foreign protein sequence is directly located on the fusion sequence after a subsequent restriction with the restriction endonucleases, followed by religation.

Description

VEKTOR-SYSTEM ZUR KLONIERUNG VON DNA VECTOR SYSTEM FOR CLONING DNA
Die Erfindung betrifft ein Vektor-System zur Klonierung.The invention relates to a vector system for cloning.
Gängige Vektoren, die zur Klonierung in prokaryontischen Systemen verwendet werden, enthalten in aller Regel folgende Merkmale: ein Selektionsgen, z.B. das für die Ampicillinre- sistenz codierende Gen, ein Markergen, das z.B. aufgrund Farbreaktion wie beim lacZ-Gen eine Unterscheidung von Vek- toren mit und ohne Insert zuläßt, und vor allem ein origin of repii ca ti on . Zum Stand der Technik ist auch auf folgende Druckschriften hinzuweisen: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. With regard to the state of the art, reference should also be made to the following publications:
a) EP 0 532 043 A2, b) EP 0 466 332 A2, c) EP 0 293 249 AI, d) GB 22 12 160 A und e) US 51 96 524.a) EP 0 532 043 A2, b) EP 0 466 332 A2, c) EP 0 293 249 AI, d) GB 22 12 160 A and e) US 51 96 524.
Das Vektorsystem gemäß lit. c besteht aus einer Sequenz von Nukleotiden, die für die Expression eines Fusionsproteins codiert. Die Fremdprotein-Sequenz ist hier allerdings unmittelbar mit der Sequenz für ein Enzym verknüpft und nicht während des Klonierungsvorgangs durch weitere Strukturen ge- trennt.The vector system according to lit. c consists of a sequence of nucleotides which codes for the expression of a fusion protein. However, 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.
In Systemen, die nicht auf Plasmiden, sondern auf Phagen basieren, kommen weitere genetische Elemente hinzu, die für die Funktionen des Lebenszyklus des Phagen wichtig sind. Wird in solchen Systemen eine fremde Sequenz in ein Marker¬ gen eingesetzt, so werden dafür gewöhnlich Schnittstellen verwendet, die im Vektor nur wenige Male bevorzugterweise nur einmal vorkommen. Eine Reihe solcher Schnittstellen ist gewöhnlich in einem sogenannten mul tiple cl oning si te angeordnet. Außer diesem mul tiple cloning si te kommen in einigen Vektoren, die der Expression fremder Proteine dienen, noch weitere Elemente hinzu. In einigen Genen wird dazu eine Se- quenz verwendet, die in einem Fusionsprotein mit dem zu klo- nierenden Insert resultiert, welches dann eine Affinität zu z.B. Maltoseresten oder Nickelchelaten hat. An diese Reste schließt sich in einigen Fällen, eine Erkennungssequenz für eine Proteinase, z.B. den Faktor Xa an. Diese Proteinase- Schnittstellen ermöglichen nach der Aufreinigung mittels Affinitätschromatographie ein Spalten des Fusionsproteins in den Affinitätsanteil und das eigentlich zu exprimierende Fremdprotein. Da jedoch im Anschluß an diese Proteinase- Schnittstelle in aller Regel eine mul tiple cl oning si te- Sequenz folgt, in die zu exprimierende Sequenz einkloniert ist, verbleiben nach dem Proteinaseprozessieren des Fusionsproteins am zu exprimierenden Fremdprotein noch weitere, oft unerwünschte Aminosäuren.In systems that are not based on plasmids but on phages, there are additional genetic elements that are important for the functions of the phage life cycle. If a foreign sequence into a marker ¬ gen used in such systems, such interfaces are used for ordinary, which is preferably in the vector only a few times only once. There are a number of such interfaces usually arranged in a so-called multiple cloning si te. In addition to this multiple cloning, other elements are added in some vectors that are used to express foreign proteins. In some genes, a sequence is used that results in a fusion protein with the insert to be cloned, which then has an affinity for, for example, maltose residues or nickel chelates. In some cases, these residues are followed by a recognition sequence for a proteinase, for example the factor Xa. After purification by means of affinity chromatography, these proteinase interfaces enable the fusion protein to be split into the affinity portion and the foreign protein which is actually to be expressed. However, since 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.
Die meisten zu exprimierenden Fremdprotein-Sequenzen liegen in einer Nukleinsäure-Umgebung vor, die eine direkte Klonierung im direkten Anschluß an eine Endoproteinase- Erkennungssequenz nicht effizient mit gängigen Klonierungs- strategien zulassen. Durch die Verwendung eines mul tiple cloning si te im Anschluß (d.h. 3' von der Endoproteinase- Erkennungssequenz) an die Endoproteinase-Erkennungssequenz wird eine Klonierung von zu exprimierenden Fremdprotein- Sequenzen sehr erleichtert. Nachteilig ist dabei, daß man auf Proteinebene nach einem Verdau mit dem die entsprechende Proteinsequenz erkennenden Enzym nicht das zu exprimierenden Fremdprotein gewinnt, sondern ein Fusionsprotein, das die zu exprimierende Fremdprotein-Sequenz umfaßt, mit weiteren Aminosäuren, die von den Resten der mul tiple cl oning si te co- diert sind .Most of the foreign protein sequences to be expressed are in a nucleic acid environment which do not allow direct cloning in direct connection with an endoproteinase recognition sequence to be carried out efficiently using conventional cloning strategies. The use of a multiple cloning site (ie 3 'from the endoproteinase recognition sequence) to the endoproteinase recognition sequence makes it much easier to clone foreign protein sequences to be expressed. The disadvantage here is that one does not obtain the foreign protein to be expressed at the protein level after digestion with the enzyme recognizing the corresponding protein sequence, but rather a fusion protein which comprises the foreign protein sequence to be expressed, with further amino acids which are derived from the residues of the multiple cl oning si te co- are dated.
Aufgabe der Erfindung ist die Bereitstellung eines neuen Vektorsystems zur Klonierung von Fremdproteinen, welches die Nachteile des Standes der Technik vermeidet. Diese Aufgabe wird durch die Merkmale der Ansprüche 1 und 10 gelöst. Vorteilhafte Ausgestaltungen ergeben sich aus den Merkmalen der Ansprüche 2 - 9 und 11.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.
Die Verwendung des hier vorgestellten Klonierungsvektor- Systems erlaubt nun, in einem nachfolgenden Verdau- und Re- ligationsschritt, die zu exprimierenden Fremdprotein-Sequenz direkt an eine Endoproteinase-Erkennungssequenz heranzuziehen. Dadurch ist gewährt, daß die zu exprimierenden Fremdprotein-Sequenz aus einem exprimierten Fusionsprotein nach Verdau mit dem die entsprechende Proteinsequenz erkennenden Enzym ohne weitere Anteile freigesetzt wird.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.
Ein besonderer Vorteil ist auch darin zu sehen, daß im Zuge des hier vorgestellten Verfahrens eine Wahl des Leserahmens frei möglich ist. Dadurch ist nicht nur ein exaktes Positionieren des zu exprimierenden Fremdgenanteils möglich, sondern auch eine Definition des Starts des Fusionsgenanteils. Um der durch den Klonierungsvorgang bedingten unerwünschten Expression zusätzlicher Peptidanteile am zu exprimierenden Fremdgen in einfacher und dennoch hochselektiver Weise vorzubeugen, wurde folgende Strategie gewählt:A particular advantage can also be seen in the fact that the reading frame can be freely selected in the course of the method presented here. This enables not only an exact positioning of the foreign gene portion to be expressed, but also a definition of the start of the fusion gene portion. In order to prevent the undesired expression of additional peptide portions of the foreign gene to be expressed caused by the cloning process in a simple yet highly selective manner, the following strategy was chosen:
Es wurde die Erkennungssequenz für das Enzym BcgI verwendet, um es innerhalb der mul tipl e clonig si te zu klonieren, in die später mit beliebigen Enzymen die Fremdsequenz eingesetzt werden kann. BcgI ist eins der Enzyme, bislang das einzig kommerziell erhältliche, die in einem definierten Ab- stand (10/12 Nukleotide) von ihrer Erkennungssequenz sowohl vor als auch hinter dieser Sequenz schneiden.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.
Damit wird durch diesen Schnitt des Enzyms also ein Sequenz- abschnitt 2x6+10+12 = 34 bp entfernt. Eine anschließende Religation erzeugt also Sequenzen, aus denen 34 Basenpaare entfernt sind. Im Fall einer gezielten Klonierung liegen also z.B. das letzte Nukleotid der Endoproteinase- Erkennungssequenz und das erste Nulkleotid des ersten Codons des zu klonierenden Proteins unmittelbar zusammen. Beispielssequenzen sind in Beispiel 1 bei der Auflistung einiger Anwendungen aufgeführt.This section of the enzyme thus removes a sequence section 2x6 + 10 + 12 = 34 bp. A subsequent religation thus generates sequences from which 34 base pairs are removed. In the case of 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.
Besondere Vorteile des BcgI-Systems umfassen dessen Neigung, je nach Pufferkonzentration in 10-50% der Fälle zu kleinen Deletionen (in der Regel 3 Nukleotide) an der Schnittstelle zu führen. Das bewirkt, daß in diesen Fällen dann die erste Aminosäure des zu exprimierenden Fremdproteins, in der Regel Methionin, nach dem Verdau mit dem die entsprechende Pro- teinsequenz erkennenden Enzym nicht mehr Bestandteil des zu exprimierenden Fremdproteins ist, das dabei freigesetzt wird.Particular advantages of the BcgI system include 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.
Man erhält ein ähnliches Ergebnis, wenn man zwei Restrikti- onsendonuklease-Erkennungsstellen verwendet; die eine im oder in unmittelbarer Nähe vom Bereich einer Endoproteinase- Erkennungssequenz und eine weitere, damit ligationskompati- ble Erkennungsstelle, unmittelbar vor der zu exprimierenden Fremdprotein-Sequenz, so daß ein Verdau mit der entsprechen- den Restriktionsendonuklease und anschließender Religation den Beginn der zu exprimierenden Fremdprotein-Sequenz unmittelbar an die Endoproteinase-Erkennungssequenz heranführt. Im folgenden wird die Erfindung anhand einiger Sequnzen und Beispiele erläutert. Es zeigenA similar result is obtained when using two restriction endonuclease recognition sites; one in or in the immediate vicinity of the region of an endoproteinase recognition sequence and a further, thus ligation-compatible recognition site, immediately before the foreign protein sequence to be expressed, so that digestion with the corresponding restriction endonuclease and subsequent religation start the expression to be expressed Foreign protein sequence leads directly to the endoproteinase recognition sequence. In the following the invention will be explained with the aid of a few sequnces and examples. Show it
Fig. 1 eine erste Sequenz,1 shows a first sequence,
Fig. 2 eine zweite Sequenz der multiplen Klonierungsstel- le,2 shows a second sequence of the multiple cloning site,
Fig. 3 eine dritte Sequenz am Beispiel BamHI,3 shows a third sequence using the example of BamHI,
Fig. 4 eine vierte Sequenz am Beispiel Clal, .Fig. 4 shows a fourth sequence using the example of Clal,.
Fig. 5 eine der vorhergehenden Sequenzen nach Restriktion und Religierung,5 shows one of the preceding sequences after restriction and religion,
Fig. 6 eine fünfte Sequenz, nämlich eine Fusionssequenz, und6 shows a fifth sequence, namely a fusion sequence, and
Fig. 7 eine sechste Sequenz, die eine Bcgl-Erken- nungsstelle enthält.7 shows a sixth sequence which contains a Bcgl recognition site.
Die in Fig. 1 gezeigte erste Sequenz läßt eine Klonierung in allen drei Leserahmen zu.The first sequence shown in Fig. 1 allows cloning in all three reading frames.
1. Liegt z.B. unmittelbar vor dem ATG eines zu exprimierenden Fremdgens eine Not I oder Ascl Erkennungsstelle, so kann dieses Enzym genutzt werden, das Fragment zu einem stumpfen Ende aufgefüllt werden und an das mit HincII geschnittene Ausgangs-Konstrukt kloniert werden. Das ist in Fig. 2 ge- zeigt.1.Is e.g. immediately in front of the ATG of a foreign gene to be expressed, a Not I or Ascl recognition site, this enzyme can be used, the fragment filled in to a blunt end and cloned to the starting construct cut with HincII. This is shown in Fig. 2.
2. Liegt z.B. unmittelbar vor dem ATG eines zu exprimieren¬ den Fremgens eine beliebige Restriktions-Erkennungsstelle, die ein 4 Nukleotide 3 'überhängendes Ende generiert, so kann dieses Enzym genutzt werden, das Fragment zu einem stumpfen Ende aufgefüllt werden und an das mit AccI geschnittene und ebenfalls aufgefüllte Ausgangs-Konstrukt kloniert werden. Das ist in Fig. 3 am Beispiel BamHI gezeigt.2. If, for example just before the ATG of the foreign gene to express ¬ any restriction recognition site, which generates an end overhanging 4 nucleotides 3 ', 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.
3. Liegt z.B. unmittelbar vor dem ATG eines zu exprimierenden Fremgens eine beliebige Restriktions-Erkennungsstelle, die ein 2 Nukleotide 3 'über überhängendes Ende generiert, so kann dieses Enzym genutzt werden, das Fragment zu einem stumpfen Ende aufgefüllt werden und an das mit Sall geschnittene und ebenfalls aufgefüllte Ausgangs-Konstrukt kloniert werden. Das ist am Beispiel Clal in Fig. 4 gezeigt.3.Is e.g. directly in front of the ATG of a foreign gene to be expressed, any restriction recognition site that generates a 2 nucleotides 3 'overhanging end, this enzyme can be used, the fragment filled up to a blunt end and at the exit cut with Sall and also filled up -Construct to be cloned. This is shown using the example of Clal in FIG. 4.
Nach einer anschließenden BcgI Restriktion und Religierung liegt in jedem der Fälle eine Sequenz vom in Fig. 5 gezeigten Typ vor.After a subsequent BcgI restriction and religion, a sequence of the type shown in FIG. 5 is present in each of the cases.
Dabei folgt das erste Nukleotid der zu exprimierenden Se- quenz unmittelbar auf die Xa Protease Erkennungsstelle.The first nucleotide of the sequence to be expressed immediately follows the Xa protease recognition site.
Beispiel 1: Design des Vektorsystems.Example 1: Design of the vector system.
Es wurde ein induzierbares prokaryontisches Promotorsystem verwendet, das mit IPTG induzierbare lacZ-System, wie es in dem Vektor pQE30 der Fa. Qiagen vorliegt. In diesem Vektor folgt danach als erste proteincodierende Sequenz eine Abfolge von 6 Histidin-Resten. In unserem Konstrukt schließt sich unmittelbar daran eine Schnittstelle für die Endoproteinase Xa an. Die entsprechende fünfte Sequenz ist in Fig. 6 gezeigt.An inducible prokaryotic promoter system was used, the lacZ system inducible with IPTG, as is present in the vector pQE30 from Qiagen. In this vector, a sequence of 6 histidine residues then follows as the first protein-coding sequence. In our construct, an interface for the endoproteinase Xa immediately follows. The corresponding fifth sequence is shown in FIG. 6.
In unserem Konstrukt folgt im Anschluß daran eine mul tiple cl oning si te, die eine BcgI-Erkennungsstelle enthält. Das ist Fig. 7 gezeigt.This is followed by a mul tiple in our construct cl oning si te that contains a BcgI recognition site. This is shown in Fig. 7.
Da eine BcgI-Stelle in dem von uns verwendeten Ausgangsvek- tor ebenfalls in der für Ampicillinresistenz codierenden Region vorkommt, wurde diese dort vorkommende Schnittstelle durch in vitro Mutagenese zerstört, die Ampicillinresistenz- Eigenschaft blieb dabei erhalten.Since a BcgI site also occurs in the starting vector we use in the region coding for ampicillin resistance, this interface there was destroyed by in vitro mutagenesis, the ampicillin resistance property was retained.
Beispiel 2: Klonierung eines Fremdproteins, hier das Strukturprotein VP1 des Polyomavirus, in dieses Klonierungsvek- tor-System.Example 2: Cloning of a foreign protein, here the structural protein VP1 of the polyomavirus, into this cloning vector system.
VP1 enthält eine für eine Klonierung geeignete Schnittstelle (BamHI) im Abstand von 6 Nukleotiden vor seinem Methionin- startkodon. Eine weitere für die Klonierung notwendige Schnittstelle, SphI, folgt im Abstand von 59 Nukleotiden auf seine codierende Sequenz. Das Klonierungsvektor-System ebenso wie das Konstrukt, das die VPl-codierende Region umfaßt, wurden mit den geeigneten Restriktionsendonukleasen, Ac- cl/blunt und SphI, geschnitten. Die codierende Sequenz wurde in das so vorbereitete Klonierungsvektorsyste ligiert, transformiert und durch Wachstum in E. coli-Zellen (XL1 blue(laclq), verhindert Expression in Abwesenheit von IPTG) amplifiziert . Größere Mengen des so hergestellten Plasmid- konstrukts wurden isoliert und aufgereinigt für die weitere Bearbeitung.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.
Das aufgereinigte Konstrukt wurde nun mit dem Enzym BcgI verdaut, in einem Agarosegel aufgetrennt und aufgereinigt und anschließend religiert und erneut in Bakterien, die für eine Expression besonders geeignet sind (RB791, ein Derivat von W3110 mit ladqLδ) transformiert und dort amplifiziert . In drei unabhängigen Klonen wurde gefunden, daß die codierende Sequenz des VPl-Proteins, beginnend mit Methionin, unmittelbar nach der letzten Aminosäure der Faktor Xa- Schnittstelle vorkam (IleGluGlyArg) .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 ladqLδ). In three independent clones it was found that the coding sequence of the VP1 protein, starting with methionine, occurred immediately after the last amino acid of the factor Xa interface (IleGluGlyArg).
In einem Klon wurde festgestellt, daß auf die Sequenz des Faktor Xa die codierende Sequenz des VPl-Proteins folgte, daß jedoch die drei Nukleotide, die für Methionin codieren, deletiert waren. Bei Wiederholung der Versuche mit weiteren Proteinen wurden ähnliche Ergebnisse erhalten.In a clone it was found that the sequence of factor Xa was followed by the coding sequence of the VP1 protein, but that the three nucleotides coding for methionine were deleted. Similar results were obtained when the experiments were repeated with other proteins.
Beispiel 3: Expression eines FremdproteinsExample 3: Expression of a foreign protein
Konstrukte, die für ein Fusionsprotein mit VPl codierten, wurden in Bakterienzellen (RB79 1) transformiert und in einer Übernachtkultur angezogen. Aus dieser Übernachtkultur wurden Bakterienkulturen bis zu einer Dichte von ca 0,8A60o herangezogen. Anschließend wurde durch Zugabe von IPTG eine Expression des Fusionsproteins umfassend die Histidinreste, die Faktor Xa-Erkennungsstelle und VPl, induziert. Nach 6 Stunden Induktion wurde Gesamtprotein geerntet und ein Aliquot zur Überprüfung der Induktionseffizienz auf ein Gel aufgetragen. Der Hauptteil dieser Proteinmischung wurde an eine Nickelchelat-Säule entsprechend den Angaben des Her- stellers (Qiagen) gebunden und dort mit verschieden Puffern gewaschen. Mittels einer Lösung, die 50 mM ETGA enthält, läßt läßt sich das Fusionsprotein aus der Säule eluieren. In unserem Fall wurde jedoch durch anschließenden Verdau unter Zugabe der Endoproteinase Faktor Xa das reine Expres- sionsprotein VPl freigesetzt. 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.

Claims

Patentansprüche claims
1. Ein Vektor-System zur Klonierung bestehend aus einer Sequenz von Nukleotiden, welches eine Fusionssequenz (1) und eine oder mehrere Restriktionsendonuklease- Erkennungsstellen für Restriktionsendonukleasen (2), die außerhalb ihrer Erkennungsstellen schneiden, zusätzlich zu einer oder mehreren weiteren für eine Klonierung eines Fremdproteins verwendbaren Restriktionsen- donuklease-Erkennungsstellen (3) sowie die Sequenz für das gewünschte Fremdprotein enthält, wobei durch anschließende Restriktion mit den Restriktionsendonukleasen (2) und nachfolgende Religierung die Fremdprotein-Sequenz unmittelbar an die Fusionssequenz (1) zu liegen kommt.1. A vector system for cloning consisting of a sequence of nucleotides, which has a fusion sequence (1) and one or more restriction endonuclease recognition sites for restriction endonucleases (2) that cut outside of their recognition sites, in addition to one or more others for cloning one Contains restriction endonuclease recognition sites that can be used for foreign protein (3) and contains the sequence for the desired foreign protein, with the subsequent restriction with the restriction endonucleases (2) and subsequent religion bringing the foreign protein sequence directly to the fusion sequence (1).
2. Vektor-System gemäß Anspruch 1, wobei die Fusionssequenz (1) eine Endoproteinase-Erkennungssequenz codiert.2. Vector system according to claim 1, wherein the fusion sequence (1) encodes an endoproteinase recognition sequence.
3. Vektor-System gemäß Anspruch 1 oder 2, wobei bei der Verwendung von mehreren Restriktionsendonulease- Erkennungsstellen (2) diese vom gleichen oder isoschizome- ren Enzym erkannt werden.3. The vector system according to claim 1 or 2, wherein when using a plurality of restriction endonulease recognition sites (2) these are recognized by the same or isoschizomeric enzyme.
4. Vektor-System gemäß Anspruch 1 oder 2, wobei die Re- striktionsendonukleasen-Erkennungsstellen (2) zwei unterschiedliche, aber ligationskompatible Restriktionsendonu- klease-Erkennungsstellen sind.4. Vector system according to claim 1 or 2, wherein the restriction endonuclease recognition sites (2) are two different, but ligation-compatible restriction endonuclease recognition sites.
5. Vektor-System gemäß Anspruch 1 oder 2, wobei nur eine Restriktionsendonuclease-Erkennungsstelle (2) mit zwei Schnittstellen vorliegt. 5. Vector system according to claim 1 or 2, wherein there is only one restriction endonuclease recognition site (2) with two interfaces.
6. Vektor-System nach Anspruch 5, wobei die Restriktions- enonuklease-Erkennungsstelle (2) eine Erkennungsstelle für BcgI ist.6. Vector system according to claim 5, wherein the restriction enonuclease recognition site (2) is a recognition site for BcgI.
7. Verwendung eines Vektor-Systems gemäß einem der Ansprüche 1-6 zur Klonierung von Fremdproteinen.7. Use of a vector system according to any one of claims 1-6 for cloning foreign proteins.
8. Kit zur Klonierung von Fremdproteinen, enthaltend ein Vektor-System gemäß einem der Ansprüche 1-6.8. Kit for cloning foreign proteins, containing a vector system according to one of claims 1-6.
9. Verfahren zur Klonierung von Fremdproteinen unter Verwendung eines Klonierungsvektor-Systems gemäß einem der Ansprüche 1-6, dadurch gekennzeichnet, daß die Nukleinsäure- Sequenz für das gewünschte Protein in eine multiple Klonie- rungsstelle (3) inseriert wird, die der Fusionssequenz (1) benachbart ist, und durch anschließende Restriktion mit der Restriktionsendonuclease (2) und nachfolgende Religierung die Fremdproteinsequenz unmittelbar an die Fusionssequenz (1) zu liegen kommt und anschließende Expression des Fusionsproteins .9. A method for cloning foreign proteins using a cloning vector system according to any one of claims 1-6, characterized in that the nucleic acid sequence for the desired protein is inserted into a multiple cloning site (3) that the fusion sequence (1st ) is adjacent, and by subsequent restriction with the restriction endonuclease (2) and subsequent religion, the foreign protein sequence comes to lie directly on the fusion sequence (1) and subsequent expression of the fusion protein.
10. Verfahren nach Anspruch 9, dadurch gekennzeichnet, daß nach der Expression des Fusionsproteins das gewünschte Pro- tein durch Spaltung des Fusionsproteins an einer am Ende der Fusionssequenz (1) lokalisierten Endoprotease-Schnittstelle erhalten wird.10. The method according to claim 9, characterized in that after the expression of the fusion protein, the desired protein is obtained by cleaving the fusion protein at an endoprotease interface located at the end of the fusion sequence (1).
11. Verfahren nach Anspruch 10, dadurch gekennzeichnet, daß durch Verwendung von BcgI als Restriktionsendonuclease (2) am gewünschten Protein die N-terminale erste Aminosäure de- letiert ist. 11. The method according to claim 10, characterized in that by using BcgI as a restriction endonuclease (2) on the desired protein the N-terminal first amino acid is deleted.
EP97953645A 1996-12-20 1997-12-18 Vectorial cloning system of dna Withdrawn EP0951539A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE19653498A DE19653498C1 (en) 1996-12-20 1996-12-20 Cloning vector system with fusion sequence
DE19653498 1996-12-20
PCT/DE1997/002963 WO1998028415A1 (en) 1996-12-20 1997-12-18 Vectorial cloning system of dna

Publications (1)

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

Family

ID=7815668

Family Applications (1)

Application Number Title Priority Date Filing Date
EP97953645A Withdrawn EP0951539A1 (en) 1996-12-20 1997-12-18 Vectorial cloning system of dna

Country Status (4)

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

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
WO2000026370A2 (en) 1998-11-03 2000-05-11 Mitokor Adenine nucleotide translocator (ant) fusion proteins and uses thereof
AU2001261518A1 (en) * 2000-05-11 2001-11-20 Mitokor Production of adenine nucleotide translocator (ant), novel ant ligands and screening assays therefor
DE10145553B4 (en) * 2001-09-16 2006-06-08 Gene Architects Ag Nucleic acid for a cloning vector
WO2005087932A2 (en) * 2003-10-03 2005-09-22 Promega Corporation Vectors for directional cloning

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 (en) * 1991-09-13 2002-04-22 株式会社日立製作所 Factor Xa linker 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
DE19653498C1 (en) 1998-07-23
WO1998028415A1 (en) 1998-07-02
US6187589B1 (en) 2001-02-13

Similar Documents

Publication Publication Date Title
DE3111405C2 (en)
DE3020528C2 (en)
DE3650783T2 (en) Use of the Apergillus glucoamylase promoter
DD209476A5 (en) METHOD FOR STABILIZING AND SELECTING HOST CELLS CONTAINING RECOMBINANT DNA
DE3023627A1 (en) MICROBIAL EXPRESSION OF QUASI-SYNTHETIC GENES
DE69731608T2 (en) IMPROVED BARSTAR GEN
DD212532A5 (en) METHOD FOR STABILIZING AND SELECTION OF HOST CELLS CONTAINING RECOMBINANT DNA
DD203330A5 (en) METHOD FOR PRODUCING HYBRID HUMAN LEUKOCYTE INTERFERONE
CH663032A5 (en) DOUBLE STRING DNA, REPLICABLE PLASMIDIC EXPRESSION CARRIERS FOR THE MICROBIAL PRODUCTION OF HYBRID HUMAN LEUCOCYTE INTERFERON AND TRANSFORMED MICROORGANISMS.
EP0616035A2 (en) Transgenic pathogen resistant organism
DD154022A5 (en) PROCESS FOR PREPARING HUMAN PRE GROWTH HORMONE
DE3537176A1 (en) Expression of a plasminogen activator in yeast
DE69532646T2 (en) PROCESS FOR THE PRODUCTION OF EXTRACELLULAR PROTEINS IN BACTERIA
DE69233245T2 (en) Process for the preparation of peptides
DE19653498C1 (en) Cloning vector system with fusion sequence
DE69627787T2 (en) Actinomycetes promoter
DE3805150A1 (en) GENE TECHNOLOGICAL METHOD FOR PRODUCING POLYPEPTIDES
DE69733540T2 (en) Plasmid
DE60033943T2 (en) SWEEP-FREE ENZYMES
DE4204103A1 (en) New gamma-amino butyric acid permease gene - for growth inhibition of microbial or plant cells or as selectable marker
EP0393039B1 (en) Process for producing peptides by specific cleavage of fusion proteins with collagenases obtained by genetic engineering
EP0822258A2 (en) Vector for positive selection, based on the cap gene, the pCAPs vector and its applications
EP1169461A2 (en) Production of pancreatic procarboxy-peptidase b, isoforms and muteins thereof, and their use
DD284691A5 (en) METHOD FOR CLONING A C-DNA SEQUENCE THAT CODES FOR AT LEAST ONE PART OF AN S-LOCUS-SPECIFIC GLYCOPROTEIN OF THE BRASSICACEAE PLANT FAMILY
AT396249B (en) METHOD FOR EXPRESSING GENES IN YEAST AND DNA FRAGMENTS AND PLASMIDES CONTAINING THESE DNA FRAGMENTS FOR USE IN THIS METHOD

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): DE FR GB

17P Request for examination filed

Effective date: 19990617

17Q First examination report despatched

Effective date: 20030411

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20040708