WO1997007220A2

WO1997007220A2 - Linker insertion mutagenesis by means of the transposition system of insertion sequence is21

Info

Publication number: WO1997007220A2
Application number: PCT/EP1996/003625
Authority: WO
Inventors: Dieter Haas; Thomas Seitz; Bernard Berger; Sergio Schmid
Original assignee: Boehringer Mannheim Gmbh; Dieter Haas; Thomas Seitz; Bernard Berger; Sergio Schmid
Priority date: 1995-08-18
Filing date: 1996-08-16
Publication date: 1997-02-27
Also published as: WO1997007220A3

Abstract

A genetic engineering process allows 4 to 11 codons to be inserted into any site of cloned genes. In genes that code for proteins, the reading pattern is not modified by an insertion. The insertions modify the protein structure, so that the proteins may be given a modified biologic activity. Transposition may be carried out according to this process both in vivo, for example in Escherichia coli, or in vitro. The process may also be used to insert epitopes.

Description

Linker insertion mutagenesis using the transposition system of the insertion sequence IS27

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. In 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). The disadvantage here is that 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. Directed in vitro mutagenesis with oligonucleotides can avoid unspecific changes in the target DNA (7,8). However, if many, randomly distributed insertions are to be isolated, this method becomes expensive and time-consuming. Linker insertion based on a transposable element (transposon, insertion sequence) which shows little specificity in the choice of the target DNA can avoid the disadvantages mentioned above. Such a system has been described for a mini-Tn5 element; Inserts of 135 bp (ie 45 codons) are thus obtained (9). Such large insertions are unsuitable for most purposes because the protein structure is interrupted too much. The method described here, which is based on the insertion sequence IS27, does not have the disadvantages mentioned.

The insertion sequence 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).

It has also been found that cointegrate formation between pME5 (or pME6) and a target plasmid can occur in vitro, in the presence of a protein extract with the overexpressed IstA and IstB proteins. The invention thus also relates to a method for the random insertion of 4 or 11 codons into any target DNA in vitro.

Both the in vivo and the in vitro method according to the invention are described in detail below.

1. In-vivo procedure

The method is based on three main components (Fig. 1):

- Escherichia coli 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.

- A "suicide" plasmid (pME6 or pME5) with an IS27-IS27 connecting sequence (= VBS) which contains restriction sites for the enzymes Sall and Bgal (FIG. 2).

- A target gene in a high copy number plasmid. z. B. pME3659 (Fig. 1). The vector used is e.g. B. pBluescript (Stratagene). The host strain ECOLIST is transformed first with the target plasmid, then with the "suicide" plasmid. Cointegrates between the two plasmids are selected (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

12 bp (8 bp from the VBS region + 4 bp target duplication) or 33 bp (29 bp from the VBS region + 4 bp target duplication) (Fig. IC). This creates insertions in the target gene that do not change the reading frame. 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.

Detailed description of the main components:

- E. coli ECOLIST is a derivative of the RR28 mutant (14). With the help of the cloning vector pLDRl 0 (15), a selectable spectinomycin / streptomycin resistance cassette of pHP45-Ω (16), the lad Q gene, the tac promoter (17) and the istA (? 45) - and B gene (13) is inserted into the E. coli αtt7? locus. The expression of the istAB genes cloned under the tac promoter can be induced with isopropyl-β-D-thiogalactoside (IPTG).

The "Suicide" plasmids pME5 and pME6. The plasmid pME5 (FIG. 2A) is a derivative of pJP5603 (18), in which a synthetic 0.14 kb oligonucleotide has been inserted as a TfcoRI-7 /.> ΚflII 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 (λpir). This strain (19) codes for the protein π which initiates the replication of the "suicide" plasmid from its oriRβK. In the absence of the protein π, 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 π-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

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 conditions for plasmid extraction, restriction, ligation and transformation are according to standard methods (23).

After the transformation of the ECOLIST strain with the target plasmid (eg pME3659), 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 μg pME5 or pME6 (23) and selected on LB plates with 50 μg canamycin / ml or 50 μg 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. In vitro method

The method is essentially based on the use of the following 3 components (Fig. 4):

- Crude extract from E. coli ED8767 with the plasmid pME3913. This plasmid (13) carries Lst.4 (P45) and is the B gene behind the inducible tac promoter.

- The "Suicide" plasmid pME5 or pME6 (see in vivo method).

- The target gene xyz in a high copy number plasmid, e.g. pBluescript (Stratagene). For example, pME3659 can be used (see in vivo method).

The cointegration between the target plasmid and pME5 (or pME6) is determined by the im

Crude extract catalyzes existing IstA (P45) and IstB proteins. Cointegrates are cleaned, introduced into E. coli ED8767 by electroporation and analyzed with restriction digestions. Insertions of pME5 (or pME6) in the target gene xyz are cut out with Sall or Bglll. After ligation, inserts of 12 bp (for SaR) or 33 bp are obtained (FIG. 4A). These are analogous to the insertions obtained in the in vivo method.

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. At these restriction sites, for example, an oligonucleotide can be used which codes for an immunologically detectable epitope (28) ("epitope mapping"). The artificially introduced Sall and ßg / II sites in the VBS region (Fig. 2B) are not the only ones possible. Because of the low recognition specificity of cointegrase, further new restriction sites in the "inverted repeats" are possible.

The following examples further illustrate the invention:

Example 1:

12 bp linker insertions in the arcB gene

With the αrc / i gene on plasmid pME3659 as the target DNA, 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.

Among the active enzymes, there were two novel properties. The insertion at position 509 (FIG. 3C) resulted in a catabolic ornithine carbamoyl transferase which could no longer be activated by the allosteric activator AMP (26). The insertion at position 990 (FIG. 3C) resulted in an enzyme that was hyperco-operative for its substrate carbamoyl phosphate (Hill coefficient n _H = 9 instead of n _H = 3 for the wild-type enzyme, at pH 6.8).

Example 2: Incubation of target DNA

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 μg 2 μl

Target plasmid DNA (e.g. pME3659) 0.25 μg 2 μl

Mix 14 μl

25mM Hepes pH 7.5 10mM M9C12

I mM dithiothreitol

50 mM KCI

1.8 mM ATP

50μg / ml bovine serum albumin

5% polyvinyl pyrrolidone K90

Crude extract (40 to 80 μg protein) 2 μl

Total volume 20 μl

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).

Legend to the figures

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. In addition to the cointegrate, 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 A: transformation of ECOLIST with pME6, selection for Cm ₅₀ medium, cointegrate formation

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. Restriction maps. The 0.14 kb fragment, designated VBS region. contains the reactive IS27-IS27 connection. CM ^R , chloramphenicol resistance, Ap ^R , ampicillin resistance. B. VBS region nucleotide sequence. It is located on the 0.14 kb Hmöttll-EcoRI fragment shown. The nucleotides mutated compared to the IS27 wild-type sequence are marked with *. Nucleotides that do not belong to the IS27 sequence are marked with °. IR: iriverted repeats.

3: 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; Θ 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.

Fig. 4: In vitro method, pME5 is shown as a suicide plasmid. A, The IstA (P45) and IstB proteins required for the reaction are overproduced using a tαc 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. In pME3913 the RBS 1 and the start codon of IstA (P46) have been deleted (13). RBS, ribosome binding site *, position on the IS27 sequence (12).

literature

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Claims

claims

1. Plasmids pME5 and pME6 with a reactive connection sequence according to the map and nucleotide sequence from FIG. 2.

2. E. coli host strain, characterized in that the istA (P45) and istB genes from IS27 are integrated and inducible in the chromosome.

3. A method for producing linker insertions by means of IS27 functions in vivo, based on (a) a plasmid according to claim 1 and (b) an E. coli host strain, which is characterized in that the istA (P45) - and is B genes from IS27 im

Chromosome are integrated and inducible.

4. Process for the production of linker inserts by means of IS27 functions in vitro, based on (a) a plasmid according to claim 1 and (b) a crude extract containing the overproduced proteins IstA (P45) and IstB from IS27.