GB2223495A - Gene encoding human basic fibroblast growth factor - Google Patents

Abstract

Synthetic DNA coding for human basic 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 Basic Fibroblast Growth Factor (bFGF).

Basic Fibroblast Growth Factor has been isolated from a variety of tissues including brain, pituitary, hypothalamus, adrenal gland, thymus, kidney, placenta and retina. This molecule is belived to play an important role in the process of angiogenesis and has been demonstrated in-vitro to be a potent mitogen and chemoattractant for capillary endothelial cells and to exert similar effects on mesodermal and neuroectodermal cells.

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 group of anionic proteins exemplified by acidic Fibroblast Growth Factor the 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 basic Fibroblast Growth Factor has been isolated and purified its amino acid sequence is reported in the literature. Comparison with Bovine bFGF shows a 99% homology in amino acid sequence suggesting a powerful selection pressure for maintainance of biological activities. It is reported that the molecule does not possess a signal sequence however it is noted that the sequence of the isolated protein lacks the first 9 amino acids of the 155 encoded by the gene.

In order to facilitate the dissection of the structure and function relationships- o-f- bFG?, its incorpo- ration- -into expression-ve-cto-rs and the p-roduction of novel chimeric proteins containitng bFGF functionality an improved novel synthetic-gene for human; bFGF is sought.

It is by no means easy to predict the design of an improved bFGF gene, since the factors that deter-mine 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 bFGF gene which is distinct from other published sequences of bFGF 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, NcoI, BspMII, HaeII, SmaI, KpnI, XbaI, BamHI and EcoRI.

According to a second aspect of the invention, there is provided DNA including the following sequence: AAG CTT ACC TGC CAT GGC AGC CGG GAG CAT CAC CAC GCT GCC CGC CCT TCC GGA GGA TGG CGG CAG CGG CGC CTT CCC GCC CGG GCA CTT CAA GGA CCC CAA GCG GCT GTA CTG CAA AAA CGG GGG CTT CTT CCT GCG CAT CCA CCC CGA CGG CCG AGT TGA CGG GGT CCG GGA GAA GAG CGA CCC TCA CAT CAA GCT ACA ACT TCA AGC AGA AGA GAG AGG AGT TGT GTC TAT CAA AGG AGT GTG TGC TAA CCG GTA CCT GGC TAT GAA GGA AGA TGG AAG ATT ACT GGC TTC TAA ATG TGT TAC GGA TGA GTG TTT CTT TTT TGA ACG ATT GGA ATC TAA TAA CTA CAA TAC TTA CCG GTC TAG AAA ATA CAC CAG TTG GTA TGT GGC ATT GAA ACG AAC TGG GCA GTA TAA ACT TGG TTC CAA AAC AGG ACC TGG GCA GAA AGC TAT ACT TTT TCT TCC AAT GTC TGC TAA GAG CTG ATA AGG ATC CGA ATT C A synthetic bFGF 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.

In particular the provision of an NcoI site including the ATG initiator codon and a favourable translation initiation sequence was provided based on the rules defined by Kozak (Kozak, N. Cell 44, 283-292 (1986)).

A further. -fea-tmre 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-pal-indromic sequence of six -base pai-rs (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 cohesiveend with a 5' extension. A suitable juxtaposition sof 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 bFGF 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 bFGF synthetic gene in accordance with the invention along with the location of useful restriction sites; Figure 4 shows the sequence of an bFGF 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 (aligome-rs); 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 BB832 and BB855 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 bFGF 495/495 duplex was cut out and extracted from the gel. The purified fragment was ligated to HinDIII/EcoRI cut DNA of the plasmid pUCl8. The ligated product was transformed into HW87 and plated on L-agar plates containing lOOmcg ml ampicillin. Colonies containing potential clones were thee grown up in L-broth containing ampicillin at lOOmcg 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 bFGF 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 Clonin 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 Biology by L.G. Davis, M.D. Dibner and J.F.

Battey publisher 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 oliucleotide 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-ioniced formamide, 10mM Tris, 10mM 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 10mien, 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 25-0 pole: of oligomer-was dried down and resuspended in 20 mcl kinase buffer (7OmM TrisepH 7.6, 10mM MgCl2, lmM ATP, 0.2 mM spermidine, 0.5mM d:ithiothreitol). 10 units of T4 polynucleotide kinase was added and the mixture incubated at 37 C for 30 mi. The kinas was then inactivated by heating at 85 C for 15 min.

4) Annealing 8 ecl 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 8.31 0.089 M boric acid, 0.25mM EDTA) containing 0.5 mcg ml ethidium bromide.

7)Isolation of the ligation product.

The band corresponding to the expected bFGF 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 equilibrate 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 bFGF 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 bFGF 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 ampicillin.

9) Isolation of plasmid DNA Plasmid DNA was prepared from the colonies containing potential bFGF 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 r -TW Any other standard cloning recipient such as HB101 would be adequate.

Claims (11)

1. DNA coding fo.r bFGF and having restriction sites for the following enzymes: HinDIII, BspMI, NcoI-, BspMII, HaeII, Sm-al; gEI, XbaI, BamHI and EcoRI.

2. DNA including the following sequence: AAG CTT ACC TGC CAT GGC AGC CGG GAG CAT CAC CAC GCT GCC CGC CCT TCC GGA GGA TGG CGG CAG CGG CGC CTT CCC GCC CGG.

GCA CTT CAA GGA CCC CAA GCG GCT GTA CTG CAA AAA CGG GGG CTT CTT CCT GCG CAT CCA CCC CGA CGG CCG AGT TGA CGG GGT CCG GGA GAA GAG CGA CCC TCA CAT CAA GCT ACA ACT TCA AGC AGA AGA GAG AGG AGT TGT GTC TAT CAA AGG AGT GTG TGC TAA CCG GTA CCT GGC TAT GAA GGA AGA TGG AAG ATT ACT GGC TTC TAA ATG TGT TAC GGA TGA GTG TTT CTT TTT TGA ACG ATT GGA ATC TAA TAA CTA CAA TAC TTA CCG GTC TAG AAA ATA CAC CAG TTG GTA TGT GGC ATT GAA ACG AAC TGG GCA GTA TAA ACT TGG TTC CAA AAC AGG ACC TGG GCA GAA AGC TAT ACT TTT TCT TCC AAT GTC TGC TAA GAG CTG 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.