GB2213821A - Synthetic gene - Google Patents

Abstract

Synthetic DNA coding for human granulocyte colony stimulating factor includes the following sequence: <IMAGE> and incorporates useful restriction sites at frequent intervals to facilitate the cassette mutagenesis of selected regions. Also included are flanking restriction sites to simplify the incorporation of the gene into any desired expression system.

Description

SYNTHETIC GENE This invention relates to synthetic genes coding for human granulocyte colony stimulating factor.

Human granulocyte colony stimulating factor (G-CSF) is one of a range of glycoprotein growth factors known as colony stimulating factors (CSFs) because they support the proliferation of haemopoietic progenitor cells. G CSF stimulates the proliferation of specific bone marrow precursor cells and their differentiation into granulocytes. It is distinguished from other CSFs by its ability to both stimulate neutrophilic granulocyte colony formation in semi-solid agar and to induce terminal differentiation of murine myelomonocytic leukemic cells in vitro. The cDNA cloning and expression of recombinant human G-CSF has been described, and it has been confirmed that the recombinant G-CSF exhibits most if not all of the biological properties of the native molecule.Sequence analysis of the cDNA clones has allowed the deduction of the amino acid sequence and reveals that the protein is 207 amino acids long with a signal sequence of 30 amino acids. The mature protein is 177 amino acids long and possesses no potential N linked glycosylation sites but several possible sites for O-linked glycosylation.

The cloning and expression of cDNA encoding human G-CSF has been described by two groups (Nagata, S. et al, Nature 319, 415-418 (1986); Souza, L.M. et al, Science 232, 61-65 (1986)). The first report of a G-CSF cDNA clone suggested that the mature protein was 180 amino acids in length. This clone when expressed in COS cells failed to generate CSF activity. The authors reported that they had also identified a cDNA clone for G-CSF that coded for a protein that lacked a stretch of three amino acids. This shorter form of G-CSF cDNA did express the expected CSF activity. The second report describes a cDNA sequence identical to this short form and makes no mention of other variants.Since these authors confirmed that the short cDNA expresses G-CSF with the expected profile of biological activity it is probable that this is the important form of G-CSF and that the longer form is either a minor splicing variant or the result of a cloning artifact.

The following patent publications relate to G-CSF: WO-A-8703689 assigned to Kirin/Amgen describes hybridomas to human G-CSF and their use in the purification of G-CSF; WO-A-8702060, assigned to Biogen, discloses human G-CSF like polypeptides and methods of producing them; WO-A-8701132, also assigned to Kirin/Amgen, discloses human G-CSF like polypeptides, sequences encoding them and methods of their production; and WO-A-8604605 and WO-A-8604506, both assigned to Chugai Seiyaku Kabushiki Kaisha, disclose a gene encoding human G-CSF and infection inhibitors containing human G-CSF.

None of the above publications relate to the production of a synthetic gene for human G-CSF.

In order to facilitate the dissection of the structure/function relationships of human G-CSF, its incorporation into expression vectors and the production of novel chimeric proteins containing G-CSF functionality an improved novel synthetic gene for human G-CSF is sought.

It is by no means easy to predict the design of an improved G-CSF gene, since the factors that determine the expressibility of a given DNA sequence are still poorly understood. Furthermore, the utility of the gene in various applications will be influenced by such considerations as codon usage and restriction sites.

The present invention relates to a synthetic G-CSF gene which is distinct from other published G-CSF genes and has advantages in the ease with which it can be modified due to the presence of useful restriction sites.

According to a first aspect of the invention, there is provided DNA coding for G-CSF and having restriction sites for the following enzymes: HinDIII, MI, APaI, BstXI, Eco47III, SacI, EcoRV, AatII, PflMI, NheI, FspI, BamHI, EcoRI According to a second aspect of the invention, there is provided DNA including the following sequence:: A AGC TTA CCT GCC ATG ACC CCC CTG GGC CCT GCC AGC TCC CTG CCC CAG AGC TTC CTG CTC AAG TGC TTA GAG CAA GTG AGG AAG ATC CAG GGC GAT GGC GCA GCG CTC CAG GAG AAG CTG TGT GCC ACC TAC AAG CTG TGC CAC CCC GAG GAG CTG GTG CTG CTC GGA CAC TCT CTG GGC ATC CCC TGG GCT CCC CTG AGC TCC TGC CCC AGC CAG GCC CTG CAG CTG GCA GGC TGC TTG AGC CAA CTC CAT AGC GGC CTT TTC CTC TAC CAG GGG CTC CTG CAG GCC CTG GAA GGG ATA TCC CCC GAG TTG GGT CCC ACC TTG GAC ACA CTG CAG CTG GAC GTC GCC GAC TTT GCC ACC ACC ATC TGG CAG CAG ATG GAA GAA CTG GGA ATG GCC CCT GCC CTG CAG CCC ACC CAG GGT GCC ATG CCG GCC TTC GCC TCT GCT TTC CAG CGC CGG GCA GGA GGG GTC CTG GTT GCT AGC CAT CTG CAG AGC TTC CTG GAG GTG TCG TAC CGC GTT CTA CGC CAC CTT GCG CAG CCC TGA TAA GGA TCC GAA TTC A synthetic G-CSF gene as described above incorporates useful restriction sites at frequent intervals to facilitate the cassette mutagenesis of selected regions. Also included are flanking restriction sites to simplify the incorporation of the gene into any desired expression system.

In particular, a BspMI site is included just upstream of the initiator ATG. 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 with DNA polymerase Klenow fragment as illustrated in Figure 2. This approach is superior to other methods of fusing genes without the initiator methionine codon since it is completely indepedent of the nature of the coding sequence.For example, the enzyme NcoI that has the recognition sequence CCATGG has been used in an analogous fashion 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 commences with a G residue. In addition, the nuclease treatment required is less reliable than the polymerase step needed for the BspMI approach. The use of BspMI sites in this way greatly facilitates 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.

Synthetic genes in accordance with the invention may be especially suitable for expression in higher eukaryotic systems, particularly mammalian cells but we would expect it to be capable of expression in other systems including E. coli, yeast and insect cells.

According to a third aspect of the invention, there is provided a genetic construct comprising DNA according to the first or second aspect or a fragment thereof.

The fragment may comprise at least 10, 20, 30, 40 or 50 nucleotides. A genetic construct in accordance with the third aspect may be a vector, such as a plasmid, cosmid or phage.

According to a fourth aspect of the invention, there is provided a process for the preparation of DNA in accordance with the first or second aspect or a genetic construct 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 second 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 shows the cDNA sequence for human G-CSF together with the deduced amino acid sequence; Figure 2 illustrates the utility of having a BspMI site preceding the gene; Figure 3a shows the sequence of a synthetic gene for human G-CSF in accordance with the invention along with the location of useful restriction sites; differences compared to -the natural cDNA sequence are indicated; Figure 3b shows a summary of the restriction sites referred to in Figure 3a; Figure 4 shows the sequence of the G-CSF synthetic gene of Figure 3 divided into oligo nucleotides; and Figure 5 shows a digrammatic summary of an exemplary assembly procedure used.

CONSTRUCTION OF THE GENE The desired gene sequence was divided into 26 oligodeoxyribo-nucleotides (oligomers) as depicted in Figure 4. The division was such as to provide 7 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 BB448 and BB473 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 550/550 bp G-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 L-broth 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 G-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 Cloning 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, lmM 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 % 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 microfilter.

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 mcl reaction and libation 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 pH8.3, 0.089 M boric acid, 0.25 mM EDTA) containing 0.5 mcg ml-l ethidium bromide.

7) Isolation of ligation product The band corresponding to the expected G-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 ul 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 EcoRI 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 G-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-l ampicillin.

9) Isolation of plasmid DNA Plasmid DNA was prepared from the colonies containing potential G-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, f.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 galU galK hsdR rpsL srl recA56 Any other standard cloning recipient such as HB101 would be adequate.

Claims (12)

1. DNA coding for G-CSF and having restriction sites for the following enzymes: HinDIII, BspMI, ApaI, BstXI, Eco47III, SacI, EcoRV, AatII, PflMI, NheI, FspI, BamHI, EcoRI

2. DNA including the following sequence: A AGC TTA CCT GCC ATG ACC CCC CTG GGC CCT GCC AGC TCC CTG CCC CAG AGC TTC CTG CTC AAG TGC TTA GAG CAA GTG AGG AAG ATC CAG GGC GAT GGC GCA GCG CTC CAG GAG AAG CTG TGT GCC ACC TAC AAG CTG TGC CAC CCC GAG GAG CTG GTG CTG CTC GGA CAC TCT CTG GGC ATC CCC TGG GCT CCC CTG AGC TCC TGC CCC AGC CAG GCC CTG CAG CTG GCA GGC TGC TTG AGC CAA CTC CAT AGC GGC CTT TTC CTC TAC CAG GGG CTC CTG CAG GCC CTG GAA GGG ATA TCC CCC GAG TTG GGT CCC ACC TTG GAC ACA CTG CAG CTG GAC GTC GCC GAC TTT GCC ACC ACC ATC TGG CAG CAG ATG GAA GAA CTG GGA ATG GCC CCT GCC CTG CAG CCC ACC CAG GGT GCC ATG CCG GCC TTC GCC TCT GCT TTC CAG CGC CGG GCA GGA GGG GTC CTG GTT GCT AGC CAT CTG CAG AGC TTC CTG GAG GTG TCG TAC CGC GTT CTA CGC CAC CTT GCG CAG CCC TGA TAA GGA TCC GAA TTC

3. A genetic construct comprising DNA as claimed in claim 1 or 2, or a fragment thereof.

A A construct as claimed in claim 3, wherein the fragment comprises at least 10 nucleotides.

5. A construct as claimed in claim 3, wherein the fragment comprises at least 20 nucleotides.

6. A construct as claimed in claim 3, wherein the fragment comprises at least 30 nucleotides.

7. A construct as claimed in claim 3, wherein the fragment comprises at least 40 nucleotides.

8. A construct as claimed in claim 3, wherein the fragment comprises at least 50 nucleotides.

9. A construct as claimed in any one of claims 3 to 8, which is a vector, such as a plasmid, cosmid or phage.

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

11. A process for the preparation of DNA as claimed in claim 1 or 2 or a genetic construct in accordance with claim 3, the process comprising coupling successive nucleotides and/or ligating appropriate oligomers.

12. DNA substantially as herein described with reference to Figure 3a.