SYNTHETIC DNA SEQUENCE CODING FOR HUMAN INSULIN-LIKE GROWTH FACTOR II

Abstract: Synthetic DNA coding for human Insulin-like Growth Factor II includes sequence (I) 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 DNA SEQUENCE CODING FOR HUMAN INSULIN-LIKE GROWTH FACTOR II This invention relates to synthetic genes coding for IGF-II. Human serum contains at least two insulin-like growth factors (IGFs) , so called because of their limited ho ology with insulin. IGF-I or somatomedin C is a mitogen that mediates the growth effects of growth hormone, predominantly throughout childhood and adolescence. The role of the related protein IGF-II is more obscure though in situ hybridisation has revealed that both IGF-I and IGF-II RNA are produced predominantly in cells of mesenchymal origin. This suggests that both IGFs may be involved in paracrine action on multiple cell types throughout development with each IGF having its own spectrum of targets. The mature form of IGF-II is 67 amino acids in length and is derived from a precursor molecule which includes a 19 amino acid signal peptide and an 89 amino acid C- ter inal extension. In addition a variant form of IGF- II has been described that possesses an extra three amino acids in the mature protein. The organisation of natural preproIGF-II is depicted in Figure 1. The amino acid sequence of IGF-II has been described (Rinderknecht and Humbel, FEBS Letters, 89, 283-286 (1978)). The cloning and expression of cDNA embodying natural sequences encoding IGF-I and IGF-II in yeast have been described by Chiron Corporation in O-A- 8600619. The cDNA cloning and sequence analysis of IGF- II including an IGF-II variant is described by Jansen, M., van Schaik, F.M.A. , van Tol, H. , van den Brande, J.L. and J.S. Sussenbach. FEBS Letters 179, 243-246 (1985) . The identification of a further variant form of IGF-II is described in Zu stein, P.P., Luthi, C. and Humbel, R. P.N.A.S 82, 3169-3172 (1985). In order to facilitate the dissection of the structure/function relationships of human IGF-II, its " incorporation into expression vectors and the " production of novel chiϊneric proteins containing IGF-II functionality an improved novel synthetic gene for human IGF-II is sought. It is by no means easy to predict the design of an improved IGF-II 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 IGF-II gene which is distinct from other published synthetic IGF-II 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/polypyri idine 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 IGF-II and having restriction sites for the following enzymes: Spill, Nsil. Ndel, Sad. PstI, BstEII, Nhβl. Xbal. PvuII, Fspl and Ba HI. According to a second aspect of the invention, there is provided DNA including the following sequence: ATG GCA TAC CGC CCG AGC GAG ACC CTG TGC GGT GGC GAG CTC GTA GAC ACT CTG CAG TTC GTT TGT GGT GAC CGT GGC TTC TAC TTC TCT CGT CCT GCT AGC CGT GTA TCT CGC CGT TCT AGA GGC ATC GTT GAA GAG TGC TGT TTC CGC AGC TGT GAT CTG GCA CTG CTC GAA ACT TAC TGC GCA ACT CCA GCA AAA TCC GAA TAA A synthetic IGF-II 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 organisms 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__a.spect 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 sequence of cDNA derived from the coding region of IGF-II RNA; Figure 2 shows a comparison of natural IGF-II coding sequence (2a) with that of an IGF-II synthetic gene (2b) in accordance with the invention; a summary of useful restriction sites is also shown; Figure 3 shows a sequence of a synthetic IGF-II gene in accordance with the invention divided into oligonucleotides; and Figure 4 shows a summary of an assembly procedure used. EXAMPLE CONSTRUCTION OF THE GENE The desired gene sequence was divided into 10 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 BB56 and BB65 were then kinased to provide them with a 5' phosphate as required for the ligation step. Complementary oligomers were then annealed and the 5 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 211/219 bp IGF-II gene duplex was cut out and extracted from the gel. The purified fragment was ligated to Sphl/BamHI cut replicative form (RF) DNA of the vector M13mpl8 (Yanisch Perron C. et al. Gene 33 103-119 (1985)). The ligated product was transformed into E. coli JM103 and plated with a lawn -of JM103 on conventional lac indicator plates. Recombinant phage were identified by the white plaques that they produced. A number of white plaques were picked and used to prepare single stranded (ss) DNA for sequence analysis. Dideoxy sequence analysis of the recombinants allowed identification of correct clones using a 17 base primer complementary to the universal primer region of M13mpl8. 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 the laboratory manual entitled Molecular Cloning by T. Maniatis, E.F. Fritsch and J. Sa brook 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 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 NH 3 . Typically, 50 g of CPG carrying 1 micromole of oligonucleotide was de-protected by incubation for 5 hr. at 70° in 600 mcl of concentrated NH 3 . 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, lOmM tris, 10 M borate, ImM EDTA, 0.1% bromophenol blue). The samples were heated at 90° 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 finall filtered through a 0.45 micron micro-filter. The concentration of purified product was measured by determining its absorbance at 260 nm. 3) inasing of oligomers 250 p ole of oligomer was dried down and resuspended in 20 mcl kinase buffer (70 mM Tris pH 7.6, 10 mM MgCl 2 , 1 mM ATP, 0.2 mM spermidine, 0.5 mM dithiothreitol) . 10 u of T4 polynucleotide kinase was added and the mixture incubated at 37° for 30 min. The kinase was then inactivated by . heating at 85° for 15 min. 4) Annealing 8 mcl of each oligomer was mixed, heated to 90° 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 MgCl 2 , 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 15° 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 meg ml "1 ethidium bromide. 7) Isolation of ligation product The band corresponding to the expected IGF-II 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 65° 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 meg 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 meg of M13mpl8 RF DNA was prepared by cleavage with SphI and BamHI as advised by the suppliers. The digested DNA was run on ah 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 IGF-II DNA ranging from 2 to 20 ng for 4 hr. using the ligation buffer described above. The ligation products were used to transform competent JM103 as- has been described. Recombinant phage were identified as those that gave white plaques when plated on a lawn of JM103 on lac indicator plates. 9} Isolation of single stranded phage DNA ss DNA was prepared from the phage giving white pla ues using published procedures. 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)). 11) Transformation Transformation was accomplished using standard procedures. The strain used as a recipient in the cloning was JM103. Any other standard cloning recipient such as HB101 would be adequate.

Claims: CLAIMS 1. DNA coding for IGF-II and having restriction sites for the following enzymes: Sphl. Nsil. Ndel. Sad. PstI. BstEII, Nhel. Xbal. PvuII. Fspl and Ba HI. 2. DNA including the following sequence: ATG GCA TAC CGC CCG AGC GAG ACC CTG TGC GGT GGC GAG CTC GTA GAC ACT CTG CAG TTC GTT TGT GGT GAC CGT GGC TTC TAC TTC TCT CGT CCT GCT AGC CGT GTA TCT CGC CGT TCT AGA GGC ATC GTT GAA GAG TGC TGT TTC CGC AGC TGT GAT CTG GCA CTG CTC GAA ACT TAC TGC GCA ACT CCA GCA AAA TCC GAA 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 claim 3, which is a vector. 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 2b.

Inventors: EDWARDS RICHARD MARK, EDWARDS, RICHARD, MARK
Applicants: BRITISH BIO TECHNOLOGY, BRITISH BIO-TECHNOLOGY LIMITED
Application year: 1988
Main classification: C12N15
IPC codes: C07K14, C12N15