GB2212160A - DNA sequence - Google Patents

Abstract

DNA comprises the sequence 5'-ACCTGCNXXXNNN-3', where each N independently represents any nucleotide and XXX represents a codon such as an initiation codon. The enzyme BspMI recognizes the sequence ACCTGC and gives rise to a staggered cut in the DNA four bases downstream of the recognition site, ie just downstream of the codon, which may be an ATG initiator. This therefore allows for the generation of a blunt end immediately following the ATG by the simple expedient of BspMI cleavage followed by repair of the cohesive end such as with DNA polymerase Klenow fragment. This approach may be used to prepare fused genes and may be found to be superior to other methods of fusing genes without the initiator methionine codon since it is completely independent of the nature of the coding sequence.

Description

DNA SEQUENCE This invention relates to certain DNA sequences and to the use of such sequences in processes for selectively cleaving DNA and preparing blunt ended DNA sequences.

In Escherichia coli and all other prokaryotes and eukaryotes, the synthesis of all proteins begins with the amino acid methionine, coded for by an initiator codon. The initiation codon is usually ATG although examples of other initiation codons such as GTG and TTG are known. The selection of a particular codon as an initiator codon depends critically on its context. For example in prokaryotes sequences 5' to the initiator ATG that are complementary to the 3' end of the 16S ribosomal provide the correct sequence environment for recognition of the ATG as an initiator codon. In eukaryotes it appears that in general the first ATG in the message is used as the initiation codon although there are additional sequence effects close to the ATG that influence the efficiency of initiation.

Therefore, whatever the host, when it is desired to express a 'foreign' gene in the host, it is necessary for an initiator sequence to be present at the 5' end of the foreign sequence.

One of the most promising applications of recombinant DNA technology is the possibility of preparing fused proteins. Although there are many examples of fused proteins which have either been prepared or proposed, one particularly useful case is the preparations of fused proteins containing a (say non-bacterial) protein of interest and a signal sequence, to enable the fused protein to be secreted into the periplasmic space or even the medium. The signal peptide is usually removed during the secretion process through the action of a leader peptidase.

One way of preparing a fused gene (that is, a gene coding for a fused protein) is to fuse two sequences (an upstream sequence and a downstream sequence) of DNA together. One sequence (such as the downstream sequence) may code for a protein of interest and the other may code for a signal peptide. Although it is possible (and in some instances advantageous) to fuse together DNA sequences having cohesive ends, the most universally applicable way of doing so is to use blunt ended double stranded DNA; two sequences of blunt ended DNA can simply be fused with T4 ligase. If the downstream sequence encoding the gene of interest is fused appropriately to the sequence encoding the signal peptide then the resultant fusion protein will be secreted and processed to liberate the desired protein devoid of extra unwanted amino acids.In particular, the fusion can be made in such a way tha the secreted protein lacks an N terminal methionine.

When designing synthetic genes for general utility, it is desirable that they can be readily adapted for expression in a range of systems. It should be possible to express them as the mature protein directly, in which case the gene will require an initiator ATG. The gene should also be readily adaptable to expression as a fusion protein. Two particular examples are fusions with a signal peptide to allow the secretion of the protein and fusion with a larger carrier protein that will stabilise the protein of interest or facilitate its purification. To be readily adaptable to all these modes of expression it is therefore desirable that the gene be provided with an initiator ATG but also be readily isolable as a DNA fragment cleaved immediately downstream of an ATG initiation site; such a fragment should for preference then be readily transformable into a blunt ended fragment.

The restriction enzyme NcoI has the recognition sequence CCATGG and has been used for this purpose since a gene can be engineered so that the initiator ATG is included in an NcoI site. Cleavage with NcoI followed by S1 or Mung Bean nuclease treatment will result in a blunt end following the ATG. This approach can only be used, however, when the codon following the ATG commneces with a G residue. In addition, the nuclease treatment required has been found to be less than reliable.

The present invention is based on a realisation that the enzyme BspMI may with advantage be used in such circumstances if a BspMI site is included just upstream of the initiator ATC. This enzyme is useful because it recognizes a non-palindromic sequence of six base pairs (5'-ACCTGC-3') and gives rise to a staggered cut in the DNA four bases downstream of the recognition site resulting in a four base cohesive end with a 5' extension. A suitable juxtaposition of the BspMI site and initiator ATG therefore allows for the generation of a blunt end immediately following the ATG by the simple expedient of BspMI cleavage followed by repair of the cohesive end such as with DNA polymerase Klenow fragment.This approach may be found to be superior to other methods of fusing genes without the initiator methionine codon since it is completely indepedent of the nature of the coding sequence. Further, an appropriately positioned BspMI site will also allow the generation of vector fragments with a blunt end immediately downstream of the sequence to which it is desired to fuse the gene. Again it is possible to design such vectors irrespective of the upstream sequence since the restriction enzyme cuts outside its recognition sequence. Combining this concept with that of generating blunt ended fragments downstream of an ATG allows the development of generic vectors for the construction of fusion proteins.

According to a first aspect of the invention, there is provided DNA comprising the sequence 5'-ACCTGCNXXXNNN3', where each N independently represents any nucleotide and XXX represents any codon, such as an initiation codon (for example ATG). Such DNA may form part of a synthetic gene, in which case it can be seen that the first aspect of the invention encompasses a synthetic gene including a site for the restriction endonuclease BspMI preceeding an initiator ATG and separated by one base, the orientation of the BspMI site being such that the enzyme cleaves the coding strand of the DNA immediately 3' to the initiator ATG.

DNA in accordance with the first aspect may be part of a genetic construct, for example a vector such as a plasmid, cosmid or phage. Such vectors are envisaged as being useful as delivery systems for downstream DNA sequences in fused genes (ie, genes coding for fused proteins).

According to a second aspect of the invention, there is provided a process for cleaving DNA immediately downstream of a codon XXX (which may be an initiation codon such as ATG), the process comprising digesting DNA comprising the sequence 5'-ACCTGCNATGNNN-3', where each N independently represents any nucleotide, with BspMI. Generally it will be desired to produce a blunt end, and so the digestion step may be followed by a step of repairing the resulting cohesive end, for example with DNA polymerase Klenow fragment. The blunt ended DNA may subsequently be fused to other DNA for example by means of T4 ligase. The invention is therefore useful for preparing fused genes. The use of BspMI sites in this way may greatly facilitate the incorporation of any synthetic or suitably modified gene into other expression sytems, in particular its fusion to a variety of secretion signals and to vectors designed for the expression of fusion proteins which include the recognition site for a specific protease such as Factor X.

In view of the fact that DNA in accordance with the first aspect may be particularly useful when incorporated within a plasmid, it will accordingly be useful to provide a suitable host vector, which may have an BspMI site or even two of them. According to a third aspect of the invention, there is therefore provided a vector, such as a plasmid, comprising two BspMI sites. Preferably, the two sites will be of opposite orientation.

According to a fourth aspect of the invention, there is provided a process for the preparation of DNA in accordance with the first aspect or a vector in accordance with the third aspect, the process comprising coupling successive nucleotides and/or ligating appropriate oligomers.

The invention also relates to other nucleic acid (including RNA) either corresponding to or complementary to DNA in accordance with the first or third aspects.

Preferred embodiments and examples of the invention will now be described. In the following description, reference is made to a number of drawings, in which: Figure 1 illustrates the utility of having a BspMI site prior to a gene; Figure 2 shows the sequence of a synthetic gene for human GM-CSF and embodying a DNA sequence in accordance with the invention along with the location of useful restriction sites; Figure 3 shows the sequence of the GM-CSF synthetic gene shown in Figure 2 divided into oligonucleotides; and Figure 4 shows a summary of an exemplary assembly procedure used.

Figure 5 shows examples of vectors including BspMI sites; Figure 6 shows an example of BspMI used to create a blunt end adjacent to a sequence encoding the signal peptide of ompA from E. coli; and Figure 7 shows an example of BspMI used to create a blunt end adjacent to a sequence encoding a fusion protein and a Factor Xa cleavage site.

EXAMPLE CONSTRUCTION OF THE GENE A GM-CSF gene sequence was divided into 18 oligodeoxyribonucleotides (oligomers) as depicted in Figure 3. The division was such as to provide seven base cohesive ends after annealing complementary pairs of oligomers. The end points of the oligomers were chosen to minimise the potential for inappropriate ligation of oligomers at the assembly stage.

The oligomers were synthesised by automated solid phase phophoramidite chemistry. Following de-blocking and removal from the controlled pore glass support the oligomers were purified on denaturing polyacrylamide gels, further purified by ethanol precipitation and finally dissolved in water prior to estimation of their concentration.

All the oligomers with the exception of the 5' terminal oligomers BB429 and BB446 were then kinased to provide them with a 5' phosphate as required for the ligation step. Complementary oligomers were then annealed and the 9 pairs of oligomers ligated together by T4 DNA ligase as depicted in Table 5. The ligation products were separated on a 2% low gelling temperature (LGT) gel and the band corresponding to the 409/409 bp GM-CSF gene duplex was cut out and extracted from the gel.

The purified fragment was ligated to EcoRI/HinDIII cut DNA of the plasmid vector pUC18. The ligated product was transformed into HW87 and plated on L-agar plates containing 100 mcg ml'l ampicillin. Colonies containing potential clones were then grown up in Lbroth containing ampicillin at 100 mcg ml-l and plasmid DNA isolated. Positive clones were identified by direct dideoxy sequence analysis of the plasmid DNA using the 17 base universal primer, a reverse sequencing primer complementary to pUC18 on the other side of the polylinker region. Some of the oligomers employed in the assembly of the gene were also used as internal sequencing primers. One GM-CSF clone was subsequently re-sequenced on both strands to confirm that no mutations were present.

METHODS All the techniques of genetic manipulation used in the manufacture of this gene are well known to those skilled in the art of genetic engineering. A description of most of the techniques can be found in one of the following laboratory manuals: Molecular Clowning by T. Maniatis, E.F. Fritsch and J. Sambrook published by Cold Spring Harbor Laboratory, Box 100, New York, or Basic Methods in Molecular Biology by L-.G.

Davis, M.D. Dibner and J.F. Battey published by Elsevier Science Publishing Co. Inc. New York.

Additional and modified methodologies are detailed below.

1) Oligonucleotide synthesis The oligonucleotides were synthesised by automated phosphoramidite chemistry using cyanoethyl phosphoramidtes. The methodology is now widely used and has been described (Beaucage, S.L. and Caruthers, M.H. Tetrahedron Letters. 24, 245 (1981)).

2) Purification of Oligonucleotides The oligonucleotides were de-protected and removed from the CPG support by incubation in concentrated NH3.

Typically, 50 mg of CPG carrying 1 micromole of oligonucleotide was de-protected by incubation for 5 hr at 700 in 600 mcl of concentrated NH3. The supernatant was transferred to a fresh, tube and the oligomer precipitated with 3 volumes of ethanol. Following centrifugation the pellet was dried and resuspended in 1 ml of water. The concentration of crude oligomer was then determined by measuring the absorbance at 260 nm.

For gel purification 10 absorbance units of the crude oligonucleotide were dried down and resuspended in 15 mcl of marker dye (90% de-ionised formamide, 10mM tris, 10 mM borate, 1mM EDTA, 0.1% bromophenol blue). The samples were heated at 900 for 1 minute and then loaded onto a 1.2 mm thick -denaturing.polyacrylamide gel with 1.6 mm wide slots. The gel was prepared from a stock of 15% acrylamide, 0.6% bisacrylamide and 7M urea in 1 X TBE and was polymerised with 0.1% ammonium persulphate and 0.025% TEMED. The gel was pre-run for 1 hr. The samples were run at 1500 V for 4-5 hr. The bands were visualised by UV shadowing and those corresponding to the full length product cut out and transferred to micro-testubes.The oligomers were eluted from the gel slice by soaking in AGEB (0.5 M ammonium acetate, 0.01 M magnesium acetate and 0.1 96 SDS) overnight. The AGEB buffer was then transferred to fresh tubes and the oligomer precipitated with three volumes of ethanol at -70 for 15 min. The precipitate was collected by centrifugation in an Eppendorf microfuge for 10 min, the pellet washed in 80 % ethanol, the purified oligomer dried, redissolved in 1 ml of water and finally filtered through a 0.45 micron micro-filter. The concentration of purified product was measured by determining its absorbance at 260 nm.

3) Kinasing of oligomers 250 pmole of oligomer was dried down and resuspended in 20 mcl kinase buffer (70 mM Tris pH 7.6, 10 mM MgCl2, 1 mM ATP, 0.2 mM spermidine, 0.5 mM dithiothreitol). 10 u of T4 polynucleotide kinase was added and the mixture incubated at 370 for 30 min. The kinase was then inactivated by heating at 850 for 15 min.

4) Annealing 8 mcl of each oligomer was mixed, heated to 900 and then slow cooled to room temperature over a period of an hour.

5) Ligation 5 mcl of each annealed pair of oligomers were mixed and 10 X ligase buffer added to give a final ligase reaction mixture (50 mM Tris pH 7.5, 10 mM MgCl2, 20 mM dithiothreitol, 1 mM ATP. T4 DNA ligase was added at a rate of 100 u per 50 ul reaction and ligation carried out at 150 for 4 hr.

6) Agarose gel electrophoresis Ligation products were separated using 2% low gelling temperature agarose gels in 1 X TBE buffer (0.094 M Tris pH 8.3, 0.089 M boric acid, 0.25 mM EDTA) containing 0.5 mcg ml1 ethidium bromide.

7) Isolation of ligation product The band corresponding to the expected GM-CSF gene ligation product was identified by reference to size markers under long wave UV illumination. The band was cut out of the gel and the DNA extracted as follows.

The volume of the gel slice was estimated from its weight and then melted by incubation at 650 for 10 min.

The volume of the slice was then made up to 400 mcl with TE (10 mM Tris pH 8.0, 1 mM EDTA) and Na acetate added to a final concentration of 0.3 M. 10 mcg of yeast tRNA was also added as a carrier. The DNA was then subjected to three rounds of extraction with equal volumes of TE equilibrated phenol followed by three extractions with ether that had been saturated with water. The DNA was precipitated with 2 volumes of ethanol, centrifuged for 10 min in a microfuge, the pellet washed in 70 % ethanol and finally dried down.

The DNA was taken up in 20 mcl of TE and 2 mcl run on a 2 % agarose gel to estimate the recovery of DNA.

8) Cloning of fragment 0.5 mcg of pUC18 DNA was prepared by cleavage with HinDIII and BamHI as advised by the suppliers. The digested DNA was run on an 0.8 % LGT gel and the vector band purified as described above.

20 ng of cut vector DNA was then ligated to various quantities of GM-CSF DNA ranging from 2 to 20 ng for 4 hr using the ligation buffer described above. The ligation products were used to transform competent HW87 as has been described. Ampicillin resistant transformants were selected on L-agar plates containing 100 mcg ml -1 ampicillin.

9) Isolation of plasmid DNA Plasmid DNA was prepared from the colonies containing potential GM-CSF clones essentially as described (Ish Horowicz, D., Burke, J.F. Nucleic Acids Research 9 2989-2998 (1981).

10) Dideoxy sequencing The protocol used was essentially as has been described (Biggin, M.D., Gibson, T.J., Hong, G.F. P.N.A.S. 80 3963-3965 (1983)). The method was modified to allow sequencing on plasmid DNA as described (Guo, L-H., Wu, R. Nucleic Acids Research 11 5521-5540 (1983).

11) Transformation Transformation was accomplished using standard procedures. The strain used as a recipient in the cloning was HW87 which has the following genotype: araD139(ara-leu)del7697 (lacIPOZY)del74 qalU galK hsdR rpsL srl recA56 Any other standard cloning recipient such as HB101 would be adequate.

Claims (20)

1. DNA comprising the sequence 5'-ACCTGCNXXXNNN-3', where each N independently represents any nucleotide and XXX represents a codon.

2. DNA as claimed in claim 1, wherein XXX represents an initiation codon.

3. DNA as claimed in claim 1 or 2, which forms part of a synthetic gene.

4. A synthetic gene including a site for the restriction endonuclease BspMI preceeding an initiator ATG and separated by one base, the orientation of the BspMI site being such that the enzyme cleaves the coding strand of the DNA immediately 3' to the initiator ATG.

5. DNA as claimed in any one of claims 1 to 4, which is part of a genetic construct.

6. DNA as claimed in claim 5, wherein the genetic construct is a vector.

7. DNA as claimed in claim 6, wherein the vector is a plasmid, cosmid or phage.

8. A process for cleaving DNA immediately downstream of a codon XXX, the process comprising digesting DNA comprising the sequence 5'-ACCTGCNXXXNNN-3', where each N independently represents any nucleotide, with BspMI.

9. A process as claimed in claim 9, wherein XXX represents an initiation codon.

10. A process for preparing a blunt ended DNA sequence, comprising preparing a DNA sequence by a process as claimed in claim 8 or 9 and subsequently repairing the resulting cohesive end.

11. A process as claimed in claim 10, wherein the cohesive end is repaired with DNA polymerase Klenow fragment.

12. A process for preparing a fused gene, comprising preparing a blunt ended DNA sequence and subsequently fusing the blunt ended DNA to other DNA.

13. A process as claimed in claim 12, wherein the fusion is carried out by means of T4 ligase.

14. A vector comprising a BspMI site.

15. A vector comprising two BspMI sites.

16. A vector as claimed in claim 15, wherein the two sites are of opposite orientation.

17. A vector as claimed in claim 14, 15 or 16 coding for a fusion protein and a factor X site.

18. A process for the preparation of DNA as claimed in any one of claims 1 to 7 or a vector as claimed in any one of claims 14 to 17, the process comprising coupling successive nucleotides and/or ligating appropriate oligomers.

19. Nucleic acid either corresponding to or complementary to DNA as claimed in any one of claims 1 to 7 or a vector as claimed in any one of claims 14 to 17.

20. DNA substantially as herein described.