GB2210618A - Synthetic gene - Google Patents

Abstract

Synthetic DNA coding for human Epidermal Growth 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 epidermal growth factor.

Epidermal growth factor (EGF) is a 53 amino acid polypeptide that in the mouse has been shown to be produced from a much larger precursor molecule. In vitro, EGF has been shown to be a powerful mitogen that stimulates the proliferation of a range of mesenchymal and epithelial cell types. The precise in vivo role of EGF has not been determined but exogenously administered EGF can elicit a range of effects including: the induction of epidermal cell growth and differentiation in new born mice, enhanced healing of skin, tendon and corneal wounds, inhibition of gastric acid secretion in dog and man, induction of lung surfactant production in premature lambs, inhibition of wool growth in sheep and restoration of sperm production in sialoadenectomised mice.

Human EGF was first purified from urine where it is present in low concentration. Sufficient EGF could be produced to allow determination of the amino acid sequence (Figure 1). This allowed the design and construction of a synthetic EGF gene and recombinant human EGF has now been produced by several groups. The recombinant material has been shown to have identical physico-chemical properties to natural murine EGF.

Urogastrone or human EGF was first identified as an activity in 1938 when it was observed that a component of human urine inhibited gastric acid secretion (Gray, J.S., Wieczorowski, E., Ivy, A.C. Science 89, 489-490 (1939)). Subsequently, epidermal growth factor from mouse submaxillary gland was purified and sequenced (Savage, C.R., Inagami, T. Cohen, S. J. Biol. Chem 247 7612-7621 (1972)). The primary sequence of human urogastrone was later determined (Gregory, H. Preston, B.M. Int. J. Peptide Protein Res. 9 107-118 (1977)) and its identity as human EGF confirmed. The chemical synthesis and cloning of a gene encoding human EGF has been described (Smith, J. et al. Nucleic Acids Research 10 4467-4482 (1982)).

The following patent publications, which relate to EGF, are known: US-A-3883497 (ICI) relates to a process for the fractionation of crude urogastrone and new beta and gamma urogastrones.

US-A-3917824 (ICI) relates to pharmaceutical compositions containing EGF or closely related derivatives thereof for inhibiting the secretion of acidic gastric juice in warm blooded animals.

US-A-3948875 (Cohen and Savage) relates to a process for the preparation of EGF and a new derivative using cross-linked polyacrylamide gel at a pH of 1-3.

JP-A-56025112 (Fuji Zoki Seiyaku) relates to the preparation of urogastrone.

NZ-A-197903 (Commw.Ind.Res.Org.) relates to depilating by administration of EGF to animals.

JP-A-57122096 (G.D. Searle) relates to a synthetic urogastrone gene, corresponding plasmid recombinants and transformed cells.

KR-A-8601588 (Hayashibara) relates to a process for the production of human EGF.

CA-A-1214739 (Amgen) relates to the manufacture and expression of genes for urogastrone polypeptides and analogues thereof.

WO-A-8404030 (Appl.Mol.Gen) relates to the manufacture and expression of genes for urogastrone polypeptides and analogues thereof.

EP-A-0097057 (Japan Chem.Res.) relates to a method of producing human EGF and pharmaceutical preparation containing it.

EP-A-0128733 (Genentech) relates to human IGF and human EGF produced from a recombinant host, a process, expression vector and recombinant host therefor, and an IGF-containing pTharmaceutical composition EP-A-0148922 (Chiron) relates to hybrid DNA synthesis of EGF.

EP-A-0131868 (Wakunaga) discloses 21-Leu human urogastrone, a corresponding gene, a corresponding recombinant plasmid, a transformed cell and a process for its production.

JP-A-60161923 (Sumitomo) discloses a microbiological preparation of human urogastrone.

JP-A-60161923 (Hitachi) relates to the preparation of human urogastrone.

EP-A-0161817 (ICI/Searle) relates generally to urogastrone.

JP-A-60248699 (Fujisawa) discloses a preparation of urogastrone.

FR-A-2566799 (Earth) discloses a gene for betaurogastrone and sub-fragments, corresponding plasmids and transformants and their preparation, and a procedure for the production of beta-urogastrone.

EP-A-0200757 (Chiron) discloses the promotion of wound healing with human EGF prepared from recombinant DNA.

AU-A-8655236 (Wellcome) relates to EGF production.

BE-A-904586 and GB-AO-8608730 (both ICI) are entitled simply "Urogastrone".

EP-A-0046039 teaches the construction of a particular synthetic gene for urogastrone. The methodology for gene synthesis, however, was in the public domain at the time as was the amino acid sequence of human EGF.

Several subsequent patent publications, as can be seen from the above summary of the prior art, disclose synthetic EGF genes as part of a process for EGF production. The concept of synthetic EGF genes is therefore known.

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

It is by no means easy to predict the design of an improved EGF gene, since the factors that determine the expressibilty 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 EGF gene which is distinct from other published synthetic EGF genes and has advantages in the ease with which it can be modified due to the presence of useful restriction sites.

When synthesising and assembling genes, problems have been encountered when there are inverted or direct repeats greater than eight bases long in the genetic sequence. In addition, areas of unbalanced base composition such as G/C or A/T rich regions or polypurine/polypyrimidine tracts have been found to lead to inefficient expression. The present invention seeks to overcome or at least alleviate these difficulties.

According to a first aspect of the invention, there is provided DNA coding for EGF and having restriction sites for the following enzymes: HindIII, NdeI, BsmI, BglI, NsiI, SphI, SacI, and BamHI According to a second aspect of the invention, there is provided DNA including the following sequence: ATG AAC AGC GAC TCT GAA TGC CCG CTG AGC CAT GAT GGC TAC TGC CTG CAC GAC GGT GTA TGC ATG TAT ATC GAA GCT CTG GAC AAA TAC GCA TGC AAC TGC GTA GTT GGT TAC ATC GGC GAA CGT TGC CAG TAC CGC GAC CTG AAA TGG TGG GAG CTC CGT TAA A synthetic EGF 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.

Codons are those that are favoured by E. coli but it is expected that the DNA would be suitable for expression in other organism including yeast and mammalian 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 or transposable element.

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 sequence of human EGF; Figure 2 shows the sequence of an EGF synthetic gene in accordance with the invention; useful restriction sites are also indicated; Figure 3 shows the sequence of a synthetic EGF gene divided into oligonucleosides; and Figure 4 shows a summary of an assembly procedure used.

EXAMPLE CONSTRUCTION OF THE GENE The desired gene sequence was divided into 8 oligodeoxyribonucleotides (oligomers) as depicted in Figure 3. The division was such as to provide 5 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 phosphoramidite 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 BB325 and BB81 were then kinased to provide them with a 5' phosphate as required for the ligation step. Complementary oligomers were then annealed and the 4 pairs of oligomers ligated together by T4 DNA ligase as depicted in Figure 4. The ligation products were separated on a 2% low gelling temperature (LGT) gel and the band corresponding to the 177/177 bp EGF gene duplex was cut out and extracted from the gel. The purified fragment was ligated to HindIII/BamHI cut DNA of the plasmid vector pUC18 (eg ATCC 37253). The ligated product was transformed into HW87 and plated on L-agar plates containing 100 mcg ml'1 ampicillin.

Colonies containing potential clones were then grown up in L-broth containing ampicillin at 100 mcg ml-1 and plasmid DNA isolated.

Positive clones were identified by direct dideoxy sequence analysis of the plasmid DNA using the 17 base universal primer. One EGF clone was subsequently resequenced on both strands to confirm that no mutations were present. Use was made of both universal primer and a new reverse sequencing primer complementary to pUC18 on the other side of the polylinker region.

METHODS All the techniques of genetic manipulation used in this Example are well known to those skilled in the art of genetic engineering. A description of most of the techniques can be found in the laboratory manual entitled Molecular Cloning by T. Maniatis, E.F. Fritsch and J. Sambrook published by Cold Spring Harbor Laboratory, Box 100, New York, USA.

Additional and modified methodologies are detailed below.

1) Oligonucleotide synthesis The oligonucleotides were synthesised by automated phosphoramidite chemistry using cyanoethyl phosrhoramidtes . 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, 1OmM 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 magnesirim 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 -700 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 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 ml-1 ethidium bromide.

7) Isolation of ligation product The band corresponding to the expected EGF 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 EGF DNA ranging from 2 to 20 ng for 4 hr using the ligation buffer described above. The ligation products were used to transform competent E.

coli 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 EGF 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 (11)

1. DNA coding for EGF and having restriction sites for the following enzymes: HindIII, NdeI, BsmI, BglI, NsiI, SphI, SacI, and BamHI

2. DNA including the following sequence: ATG AAC AGC GAC TCT GAA TGC CCG CTG AGC CAT GAT GGC TAC TGC CTG CAC GAC GGT GTA TGC ATG TAT ATC GAA GCT CTG GAC AAA TAC GCA TGC AAC TGC GTA GTT GGT TAC ATC GGC GAA CGT TGC CAG TAC CGC GAC CTG AAA TGG TGG GAG CTC CGT TAA

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

4. 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, phage or transposable element.

10. 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.

11. DNA substantially as herein described with reference to Figure 2.