GB2223496A - Gene encoding human acidic fibroblast growth factor - Google Patents

Abstract

Synthetic DNA coding for human acidic fibroblast 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 mature Human Acidic Fibroblast Growth Factor (aFGF).

Acidic Fibroblast Growth Factor is a member of a group of anionic endothelial cell molecules including a and 6 Endothelial Cell Growth Factors, Eye Derived Growth Factor and a-retina-derived Growth Factor. The role of these molecules in the regulation of endothelial cell activity is as yet only partly understood. It is suggested that along with a cationic group of proteins exemplified by basic Fibroblast Growth Factor the anionic molecules act in a 'cascade' effect to control endothelial cell activity either co-ordinately through synergistic effects or via independent routes.

The regulation of endothelial cells is essential to the protection of arteries, veins and capillaries from the effect of thrombogenesis. Endothelial cells do not show cell division except in the case of wounding of blood vessels, their stimulation and control by these factors is also thought to be important in the development of tumours and the process of atherosclerosis.

Human acidic Fibroblast Growth Factor has been isolated and purified its amino acid sequence is reported in the literature. Based on comparisons with the bovine molecule of the same type a relationship with Endothelial Cell Growth Factor in each case has also been suggested. It is reported that processing of Endothelial Cell Growth Factor through the cleavage of the first 15 amino acids generates aFGF in each case.

Since there may be specific biological activities associated with each particular molecule it is sought in this application to provide a unique DNA sequence encoding aFGF to allow expression of the molecule and analysis of its function in relation to its precursor.

In the assembly strategy of aFGF advantage is taken of the relationship with its precursor 6 Endothelial Cell Growth Factor to permit modification and subcloning of a unique sequence by means of specifically synthesised oligonucleotides to provide and in frame 5' amino acid sequence.

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

It is by no means easy to predict the design of an improved aFGF 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 aFGF gene which is distinct from other published sequences of aFGF genes and has advantages in the ease with which it can be modified due to the prescence 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 DNA sequence. In addition, areas of unbalenced 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 aFGF and having restriction sites for the following enzymes: HinDIII, BspMI, AvaI, PstI, BspMII, PvuII, ScaI, SphI, Ural, BamHI and EcoRI.

According to a second aspect of the invention, there is provided DNA including the following sequence: AAG CTT ACC TGC CAT GTT TAA TCT GCC TCC CGG GAA TTA CAA GAA GCC CAA ACT CCT CTA CTG CAG CAA CGG GGG CCA CTT CCT GAG GAT TCT TCC GGA TGG CAC AGT GGA TGG GAC AAG GGA CAG GAG CGA CCA GCA CAT TCA GCT GCA ACT CAG TGC GGA AAG CGT GGG GGA GGT GTA TAT AAA GAG TAC CGA GAC TGG CCA GTA CTT GGC AAT GGA CAC CGA CGG GCT TTT ATA CGG CTC ACA GAC ACC AAA TGA GGA ATG TTT GTT CCT GGA AAG GCT GGA GGA GAA CCA TTA CAA CAC CTA TAT ATC CAA GAA GCA TGC AGA GAA GAA TTG GTT TGT TGG CCT CAA GAA GAA TGG GAG CTG CAA ACG CGG TCC TCG GAC TCA CTA TGG CCA GAA AGC AAT CTT GTT TCT CCC CCT GCC AGT CTC TTC TGA TTA ATA AGG ATC CGA ATT C A synthetic aFGF gene as described above incorporates useful restriction sites at frequent intervals to facilitate the cassette mutagenesis of selected regions.

Also included are flanking sites to simplify the incorporation of the gene into any desired expression system.

A further feature of the design is the inclusion of a BspMI site just upstream of the initiator ATG. This enzyme is useful because it recognizes a non-palindromic sequence of six base pairs (S'-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 commneces 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 will 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.

Synthetic genes in accordance with the invention are generally designed primarily for expression in higher eukaryotic systems, particularly mammalian cells but they are expected 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 aspest 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 a cDNA sequence for aFGF together with deduced amino acid sequence; Figure 2 illustrates the utility of having a BspMI site preceding the gene; Figure 3 shows the sequence of an aFGF synthetic gene in accordance with the invention along with the location of useful restriction sites; Figure 4 shows the sequence of an aFGF gene divided into oligonucleotides; and Figure 5 shows a summary of an exemplary assembly procedure.

EXAMPLE CONSTRUCTION OF THE GENE The desired gene sequence was divided into 24 oligodeoxribonucleotides (oligomers) as depicted in Figure 4.

The division was such as to provide seven base cohesive ends after annealing complementary pairs of oligiomers.

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 oligimers BB768 and BB791 were then kinased to provide them with a 5' phosphate as required for the ligation step. Complementary oligomers were then annealed and the pairs of oligomers ligated together by T4 DNA ligase as depicted in Figure 5. The ligation products were separated on a 2% low gelling temperature (LGT) gel and the band corresponding to the precursor aFGF 495/495 duplex was cut out and extracted from the gel.

The purified fragment was ligated to HinDIII/EcoRI cut DNA of the plasmid put18. The ligated product was transformed into How87 and plated on L-agar plates containing 100mcg ml ampicillin. Colonies containing potential clones were then growl up in L-broth containing ampicillin at 100mcg ml and plasmid DNA isolated. Positive clones were identified by direct di-deoxy sequence analysis of the plasmid DNA using the 17 base universal primer and reverse sequencing primer complementary to pUC18 on each side of the polylinker region. To determine the sequence of certain internal regions oligomers used in the construction were also employed as sequencing primers.One clone of precursor aFGF was subsequently re-sequenced on both strands to confirm that no mutations were present.

Following the identification of this correct clone plasmid DNA was isolated and the DNA digested using HinDIII and XmaI. The larger fragment containing the original plasmid sequence together with the majority of the cloned insert was isolated using LT agarose as described previously. A linker composed of the annealed oligonucleotides BB829 & BB830 providing HinDIII and XmaI ends encompassing the correct 5'amino acid sequence was inserted as descibed above.

The isolation of a clone encoding the correct sequence for mature aFGF was performed in the manner descibed earlier following the methods described for transformation and plasmid sequencing.

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 USA, or Basic Methods in Molecular Biolo by L.G. Davis, M.D. Dibner and J.F.

Battey published by Elsevier Science Publishing Co. Inc.

New York, USA.

Additional and modified methodologies are detailed below.

1) Oligonucleotide synthesis The oligonucleotides were synthesised by automated phosphoramidite chemistry using cyanoethyl phosphoramidites. 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 deprotected and removed from the CPG support by incubation in concentrated NH3.

Typically, 50mg of CPG carrying 1 micromole of oligogucleotide was deprotected by incubation for Shrs at 70 C in 600 mcl of concentrated NH3. The supernatant was transfered 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 oligonucleotide were dried down and resuspended in 15 mcl of marker dye (90% de-ionised formamide, 1OmM Tris, l0mM borate, lmM EDTA, 0.1% bOomophenol blue).

The samples were heated at 90 C for lmin and then loaded onto a 1.2mm thick denaturing polyacrylamide gel with 1.6mm 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 volts for 4-5 hrs.

The bands were visualised by UV shadowing and those corresponding to the full length product were cut out and transfered to micro-testubes. The oligomers were were eluted from the gel slice in AGEB (0.5 M ammonium acetate, 0.01 M magnesium acetate and 0.1% SDS) overnight. The AGEB buffer was then transfered to fresh tubes and the oligomer precipitated with three volumes of ethanol at -70 C for 15min. The precipitate was collected by centrifugation in an Eppendorf microfuge for 10mint the pellet washed in 80% ethanol, the purified oligomer dried , redissolved im lml 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 (70mM Tris pH 7.6, 10mM MgCl2, lmM ATP, 0.2 mM spermidine, 0.5mM dithiothreitol). 10 units of T4 polynuclegtide kinase was added and the mixture incubated at 37 C for 30 min. The kinase was then inactivated by heating at 85 C for 15 min.

4) Annealing 8 mOcl of each pair of oligomer was mixed, heated to 90 C and then slow cooled to room temperature over a period of 1 hr.

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 (50mM Tris pH 7.5, 10mM MgCl , 20mM dithiothreitol, lmM ATP). T4 DNA ligase was aided at a rate of 100 units per 50 mcl reaction and ligation was carried out at 15 C 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, 0.25mM EDTA) containing 0.5 mcg ml 1 ethidium bromide.

7)Isolation of the ligation product.

The band corresponding to the expected aFGF 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 was estimated from its weight and then melted by incubation at 65 C for 10mien. The volume of the slice was then made up to 400 mcl with TE (l0mM Tris pH 8.0, lmM EDTA) and Na acetate added to a final concentration of 0.3 M. 10 mcg of yeast tRNA was also added as carrier. The DNA was the subjected to three rounds of extraction with equal volumes of TE equilibrated phenol followed by three extractions with water saturated ether. The DNA was precipitated with two volumes of ethanol, centrifuged for 10mien in a microfuge, the pellet washed in 70% ethanol and finally dried down. The DNA pellet was taken up in 20 mcl of TE and 2 mcl of this volume were 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 aFGF DNA ranging from 2 to 24 ng for 4 hr using the ligation buffer described above.

20ng of cut vector DNA was then ligated to various quantities of aFGF DNA ranging from 2 to 20 ng for 4 hr using the ligation buffer described above. The ligation products were used to transform competent HOWS7 as has been described. Ampicillin resistant transformants were selected on L-agar plates containing 100 mcg ml ampicillin.

9) Isolation of plasmid DNA Plasmid DNA was prepared from the colonies containing potential aFGF 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 recipient in the cloning was HW87 which has the following genotype: araD139(ara-leu)del7697 (lacIPOZY)del74 galU galK hsdR rpsL srl recATW Any other standard cloning recipient such as HB101 would be adequate.

Claims (11)

1. DNA coding for aFGF and having restriction sites for the following enzymes: HinDIII, BspMI, AvaI, PstI, BspMII, PvuII, ScaI, SphI, Balm, BamHI and EcoRI.

2. DNA including the following sequence: AAG CTT ACC TGC CAT GTT TAA TCT GCC TCC CGG GAA TTA CAA GAA GCC CAA ACT CCT CTA CTG CAG CAA CGG GGG CCA CTT CCT GAG GAT TCT TCC GGA TGG CAC AGT GGA TGG GAC AAG GGA CAG GAG CGA CCA GCA CAT TCA GCT GCA ACT CAG TGC GGA AAG CGT GGG GGA GGT GTA TAT AAA GAG TAC CGA GAC TGG CCA GTA CTT GGC AAT GGA CAC CGA CGG GCT TTT ATA CGG CTC ACA GAC ACC AAA TGA GGA ATG TTT GTT CCT GGA AAG GCT GGA GGA GAA CCA TTA CAA CAC CTA TAT ATC CAA GAA GCA TGC AGA GAA GAA TTG GTT TGT TGG CCT CAA GAA GAA TGG GAG CTG CAA ACG CGG TCC TCG GAC TCA CTA TGG CCA GAA AGC AAT CTT GTT TCT CCC CCT GCC AGT CTC TTC TGA TTA ATA AGG ATC CGA ATT C

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 or phage.

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 2a.