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WO1997007220A2 - Mutagenese par insertion de lieurs au moyen du systeme de transposition de la sequence d'insertion is21 - Google Patents

Mutagenese par insertion de lieurs au moyen du systeme de transposition de la sequence d'insertion is21 Download PDF

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Publication number
WO1997007220A2
WO1997007220A2 PCT/EP1996/003625 EP9603625W WO9707220A2 WO 1997007220 A2 WO1997007220 A2 WO 1997007220A2 EP 9603625 W EP9603625 W EP 9603625W WO 9707220 A2 WO9707220 A2 WO 9707220A2
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WO
WIPO (PCT)
Prior art keywords
plasmid
target
pme6
pme5
sequence
Prior art date
Application number
PCT/EP1996/003625
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German (de)
English (en)
Other versions
WO1997007220A3 (fr
Inventor
Dieter Haas
Thomas Seitz
Bernard Berger
Sergio Schmid
Original Assignee
Boehringer Mannheim Gmbh
Dieter Haas
Thomas Seitz
Bernard Berger
Sergio Schmid
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 Boehringer Mannheim Gmbh, Dieter Haas, Thomas Seitz, Bernard Berger, Sergio Schmid filed Critical Boehringer Mannheim Gmbh
Publication of WO1997007220A2 publication Critical patent/WO1997007220A2/fr
Publication of WO1997007220A3 publication Critical patent/WO1997007220A3/fr

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    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1018—Carboxy- and carbamoyl transferases (2.1.3)

Definitions

  • the invention relates to plasmids, corresponding host cells and a genetic engineering method which allows insertions of 4 to 11 codons into cloned genes at any point.
  • genes that code for proteins the reading frame is not changed by an insertion. Rather, the inserts modify the protein structure, which means that proteins can receive an altered biological activity.
  • the transposition process of the method can take place in vivo, for example in Escherichia coli, or in vitro. The method can also be used to insert epitopes.
  • Linker insertion mutagenesis (in-frame insertion mutagenesis; codon cassette mutagenesis) can serve to elucidate structure-function relationships in proteins.
  • the insertion of 2, 3, 4 or more codons into genes which code for proteins causes the protein structure to be modified. This can have various consequences: loss of the biological function of the protein, change in the stability or activity of the protein, etc. Under certain circumstances, an insertion does not lead to any detectable change in the target protein (1).
  • Previously known methods of linker insertion mutagenesis are mostly based on the insertion of oligonucleotides or selectable cassettes into the target DNA.
  • the oligonucleotides or cassettes used generally carry a restriction site.
  • Naturally occurring recognition sites for restriction enzymes in the target DNA can be used for linker insertion; in this case the target DNA is opened in vitro with a restriction enzyme (2,3,4).
  • the disadvantage of this method is that the insertion sites are not randomly distributed. This limitation can be eliminated with another method: DNase I in lower Concentration and in the presence of Mn 2+ causes double-strand breaks in DNA at any sites (1.5).
  • DNase I can often delete several nucleotides near the break site, so that the target DNA along with the desired one Insertion may have further undesired changes (6). This means that many insertion constructs have to be sequenced and that many of them may prove unsuitable.
  • IS27 can spontaneously manifest tandem duplications in various bacteria [here designated (IS27) 2 ]. Plasmids with (IS27) 2 form cointegrates together with other plasmids; in E. coli the cointegrate frequency can reach 10 "1 (10, 11). IS27 carries 2 genes, istAB, which are organized as an operon. Their nucleotide sequence is known (12). The istA gene encodes two proteins, which are expressed in the same reading frame and are distinguished by different N-termini: transposase (approx. 46 kDa) and co-integrase (approx. 45 kDa) (10, 11).
  • the B gene codes for a protein with an auxiliary function; without this, the transposition frequency is greatly reduced (10, 11.)
  • the formation of cointegrates takes place in several steps: first, the (IS27) 2 donor plasmid in the IS27-IS27 connection sequence is opened by cutting at the 3 'ends of the IS elements are set (11). The (IS27) 2 plasmid processed in this way is inserted into the target plasmid by means of a non-replicative mechanism. A target duplication of 4 (rarely 5 ) bp (12). It has now been found that the IS27-IS27 connection sequence can be artificially changed at various points. This creates recognition sites for restriction enzymes without significantly influencing cointegrate formation.
  • the present invention thus relates to a method for the random insertion of 4 or 11 codons in any target DNA in a specially constructed mutant of Escherichi coli and with the aid of two plasmids (pME5, pME6).
  • the invention thus also relates to a method for the random insertion of 4 or 11 codons into any target DNA in vitro.
  • the method is based on three main components (Fig. 1):
  • ECOLIST A host strain with chromosomally integrated istA (P45) - and istB genes which are under the control of the tac promoter and the Lacl repressor.
  • FIG. 1A Insertions of the "suicide" plasmid into the chromosome of the host strain occur, but are rare.
  • Target plasmid and cointegrate can co-exist in the cell for a long time (FIG. 1A).
  • the cointegrate is purified by renewed transformation of any strain of E. coli with diluted plasmid DNA (FIG. IB).
  • the cointegrates are examined with restriction analysis; Insertions of the "suicide" plasmid in the target gene are examined further, insertions in the vector part are discarded.
  • the cointegrates of interest are restricted in vitro with Sall or Bglll. Most of the "suicide" plasmid is lost in the ligation, and an insertion of remains
  • the plasmid restricted and ligated with Sall or with BglII is introduced into E. coli by transformation and can be used for the expression of the mutated target gene.
  • E. coli ECOLIST is a derivative of the RR28 mutant (14).
  • a selectable spectinomycin / streptomycin resistance cassette of pHP45- ⁇ (16) the lad Q gene, the tac promoter (17) and the istA (? 45) - and B gene (13) is inserted into the E. coli ⁇ tt7? locus.
  • the expression of the istAB genes cloned under the tac promoter can be induced with isopropyl- ⁇ -D-thiogalactoside (IPTG).
  • the "Suicide" plasmids pME5 and pME6 are a derivative of pJP5603 (18), in which a synthetic 0.14 kb oligonucleotide has been inserted as a TfcoRI-7 /.> ⁇ flII fragment.
  • the central region is a modified IS27-IS27 connection sequence, which restriction sites for Bgäl and Sall in the "inverted repeats" from IS27 carries (Fig. 2B).
  • the plasmid can be replicated in the auxiliary strain E. coli CCI 18 ( ⁇ pir). This strain (19) codes for the protein ⁇ which initiates the replication of the "suicide" plasmid from its oriR ⁇ K. In the absence of the protein ⁇ , this plasmid cannot be replicated.
  • the plasmid pME6 (FIG. 2A) is a derivative of pGP704 (19) with a chloramphenicol resistance cassette from Tn7725 (20) and the VBS region (FIG. 2B).
  • the principle of protein ⁇ -dependent replication is the same as for pME5.
  • the target plasmid pME3659 (Fig. 1) is shown as an example.
  • the vector is said to be high
  • pBluescript (21) Possess number of copies; pBluescript (21) is suitable.
  • the target gene is, for example, the arcB gene from Pseudomonas aeruginosa (22).
  • the arcB gene encodes catabolic ornithine carbamoyl transferase (22).
  • the culture of this strain is diluted 1: 100 in LB medium (23) and 1 mM IPTG is added. The culture is grown at 37 ° C to OD 550 ⁇ 0.5.
  • the cells are transformed with 0.3 ⁇ g pME5 or pME6 (23) and selected on LB plates with 50 ⁇ g canamycin / ml or 50 ⁇ g chloramphenicol / ml. Typically, about 50 colonies resistant to kanamycin or chloramphenicol are obtained; the majority of these contain a cointegrate, in addition to the target plasmid (FIG. 1A).
  • Plasmid preparations produced according to the CTAB method (24) are diluted 1: 100 to 1: 1000 and used for the transformation of E. coli ED8767 (25). By selecting for cointegrates (kanamycin or chloramphenicol), these can be purified from the target plasmid. 2.
  • cointegrates kanamycin or chloramphenicol
  • the method is essentially based on the use of the following 3 components (Fig. 4):
  • This plasmid (13) carries Lst.4 (P45) and is the B gene behind the inducible tac promoter.
  • the target gene xyz in a high copy number plasmid e.g. pBluescript (Stratagene).
  • pME3659 can be used (see in vivo method).
  • the cointegration between the target plasmid and pME5 (or pME6) is determined by the im
  • the methods according to the invention can be used in particular for the generation, in vivo or in vitro, of new Safl or ßg / II restriction sites in any target genes.
  • an oligonucleotide can be used which codes for an immunologically detectable epitope (28) ("epitope mapping").
  • epitope (28) immunologically detectable epitope 228
  • the 12 bp insertions shown in FIGS. 3A, B were obtained.
  • the "suicide" plasmid was pME5 or pME6.
  • the position of each insertion was determined by nucleotide sequence analysis. A comparison of the insertion sites obtained showed no homology; there is no evidence that the IS27 transposition system prefers a particular target sequence (12). 45% of the insertion mutants retained enzymatic activity; 55% had lost activity.
  • the insertion at position 509 resulted in a catabolic ornithine carbamoyl transferase which could no longer be activated by the allosteric activator AMP (26).
  • the crude extract of E. coli ED8767 / pME3913 is produced according to the Schmid method (13).
  • the incubation mixture is composed of the following components on ice: pME5 (or pME6) DNA 0.25 ⁇ g 2 ⁇ l
  • Target plasmid DNA e.g. pME3659 0.25 ⁇ g 2 ⁇ l
  • the reaction mixture is incubated at 30 ° C for 60 minutes. Thereafter, the ratio of cointegrates to target plasmid is approximately IO "3.
  • Total plasmid DNA (24) is cleaned once with phenol / chloroform (23) and then with the Geneclean II kit (BIO 101). Cleaned Plasmid DNA is introduced into E. coli ED8767 by electroporation (27), with selection for the kanamycin resistance of pME5 (or the chloramphenicol resistance of pME6).
  • Fig. 1 In vivo method; Selection, cleaning and dissolution of coin integrates. The most important restriction interfaces are given.
  • the cointegration takes place after the first transformation.
  • the target plasmid is also present in cells A.
  • After the second transformation with diluted plasmid DNA B contains only one cointegrate.
  • SS suicide plasmid, eg pME6; l ] target plasmid, e.g. B. pME3659.
  • CM R chloramphenicol resistance.
  • Step after B isolation of plasmid DNA, dilution of the DNA IO "2 to IO " 3 and transformation, selection on Cm 50 medium
  • Step after C isolation of plamsid DNA, restriction with Safl or BgRl, ligation, transformation
  • Fig. 2 VBS plasmids pME5 and pME6.
  • A localization of the insertions of pME5 and pME6 in arcB gene.
  • the positions refer to the first nucleotide of the ctrC-5 fragment (22).
  • the arrows indicate the orientation, which is defined in B. ⁇ ? ⁇ Start of arcB translation; ⁇ End of arcB translation.
  • B definition of orientation, after the integration of pME5 and pME6 in arcB ( 1 --- 2 ).
  • C sequences of insertions at position 509 and 990. Insertion 509 was made with pME6 and insertion 990 with pME5.
  • the orientation of the insertions is defined in B.
  • the target duplications are underlined.
  • pME5 is shown as a suicide plasmid.
  • A The IstA (P45) and IstB proteins required for the reaction are overproduced using a t ⁇ c promoter which has been cloned in front of the IS27 genes. Ap, ampicillin resistance.
  • B pME3913 is used for the overproduction of the IS27 proteins. The sequence deviating from the IS27 wild type sequence is given below.
  • RBS ribosome binding site *, position on the IS27 sequence (12).

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Abstract

Le procédé de génie génétique décrit permet d'insérer de 4 à 11 codons dans n'importe quel site de gènes clonés. Dans des gènes qui codent des protéines le schéma de lecture n'est pas modifié par une insertion. Les insertions modifient la structure des protéines, ce qui peut conférer aux protéines une activité biologique modifiée. La transposition réalisée selon ce procédé peut se faire in vivo, par exemple dans Escherichia coli, ou in vitro. Le procédé peut également être appliqué pour l'insertion d'épitopes.
PCT/EP1996/003625 1995-08-18 1996-08-16 Mutagenese par insertion de lieurs au moyen du systeme de transposition de la sequence d'insertion is21 WO1997007220A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CH237395 1995-08-18
CH2373/95-3 1995-08-18

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WO1997007220A2 true WO1997007220A2 (fr) 1997-02-27
WO1997007220A3 WO1997007220A3 (fr) 1997-03-20

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6686713B2 (en) 2001-09-26 2004-02-03 S.N.R. Roulements Pulse-controlled electric power-assisted steering system

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BACTERIAL CONJUGATION (CLEWELL, D., ed.), PLENUM PRESS, New York, 1993; Chapter 6; REIMMANN, C. & HAAS, D.:'Mobilization of chromosomes and nonconjugative plasmids by cointegrative mechanisms.' XP000616906 in der Anmeldung erw{hnt *
Diss. ETH Nr. 10197. Z]RICH 1993. SCHMID, S.:'Die Insertionssequenz IS21: Entwicklung eines In-vitro Integrationssystems' XP002024154 in der Anmeldung erw{hnt *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, Bd. 88, Juni 1991, WASHINGTON US, Seiten 5457-5461, XP002024153 HOEKSTRA, M. ET AL.: "A Tn3 derivative that can be used to make short in-frame insertions within genes" in der Anmeldung erw{hnt *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6686713B2 (en) 2001-09-26 2004-02-03 S.N.R. Roulements Pulse-controlled electric power-assisted steering system

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