GB2210883A - Synthetic gene - Google Patents

Abstract

Synthetic DNA coding for human interleukin 3 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 interleukin 3.

Human interleukin 3 (IL3) is one of a range of growth factors known as colony stimulating factors (CSFs) because they support the proliferation of haemopoietic progenitor cells. In the mouse, IL3 has a broad spectrum of activity and is known to act on early multipotent progenitor cells. The gene for a human homologue of murine IL3 has been described, and it is likely that this CSF will exhibit a broad activity similar to that of murine IL3. The similarity between human and mouse IL3 is less than that observed for other CSFs, however, and this may indicate that the in vivo function of IL3 may have diverged somewhat.

Using the sequence of the human and mouse genes and by analogy with the cDNA sequences of murine and gibbon 1L3 it has proved possible to deduce the cDNA sequence and thus the amino acid sequence of human IL3. It appears that human IL3 is expressed as a 152 amino acid precursor with an N-terminal signal peptide of 19 amino acids that is cleaved in the secretion process to give a mature IL3 of 133 amino acids and ala-pro as the Nterminus.

The first IL3 to be identified was the murine protein, identified by its ability to induce a specific steroid dehydrogenase in splenic lymphocytes from nude mice (Ihle, J.H. et al. Immunol. Res. 63, 5-32 (1982)). The cloning of murine IL3 and its expression in a prokaryotic host is disclosed in WO-A-8502863 (Australian National University and Biotechnology Australia Pty Ltd). The cloning of the gene for human IL3 and the deduction of the cDNA and amino acid sequence heve been described (Yang, Y-C. et al. Cell 47, 3-10 (1986)).

In order to facilitate the dissection of the structure/function relationships of human IL3, its incorporation into expression vectors and the production of novel chimeric proteins containing IL3 functionality an improved novel synthetic gene for human IL3 is sought.

It is by no means easy to predict the design of an improved IL3 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 IL3 gene which is distinct from other published synthetic IL3 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 IL3 and having restriction sites for the following enzymes: HindIII, NcoI, SpeI, HpaI, ClaI, Nsp(7524)V, NsiI, BalI, EagI, XmnI, EcoRI, BspMII, FspI, NheI, BamHI and EcoRI.

According to a second aspect of the invention, there is provided DNA including the following sequence: ATG GCT CCC ATG ACC CAG ACA ACT AGT TTG AAG ACA AGC TGG GTT AAC TGC TCT AAC ATG ATC GAT GAA ATT ATA ACA CAC TTA AAG CAG CCA CCT TTG CCT TTG CTG GAC TTC AAC AAC CTC AAT GGG GAA GAC CAA GAC ATT CTG ATG GAA AAT AAC CTT CGA AGG CCA AAC CTG GAG GCA TTC AAC AGG GCT GTC AAG AGT TTA CAG AAT GCA TCA GCA ATT GAG AGC ATT CTT AAA AAT CTC CTG CCA TGT CTG CCC CTG GCC ACG GCC GCA CCC ACG CGA CAT CCA ATC CAT ATC AAG GAC GGT GAC TGG AAT GAA TTC CGG AGG AAA CTG ACG TTC TAT CTG AAA ACC CTT GAG AAT GCG CAG GCT CAA CAG ACG ACT TTG AGG CTA GCG ATC TTT TAG A synthetic IL3 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.

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 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 a deduced cDNA sequence for human IL3; Figure 2 shows a sequence of a synthetic gene for human IL3 along with location of useful restriction sites; Figure 3 shows a sequence of the IL3 synthetic gene divided into oligonucleotides; and Figure 4 shows a summary of the assembly procedure used.

The design for the synthetic mature human IL3 gene was based on the deduced cDNA sequence but with modifications to incorporate useful restriction sites to facilitate the cassette mutagenesis of selected regions. Also included were flanking restriction sites to simplify the incorporation of the gene into any desired expression system. In particular, an NcoI site was engineered to encompass the ATG initiator codon and a favourable translation initiation sequence was provided based on the rules defined by Kozak (Kozak, N.

Cell 44, 28-3-292 (1986)). A SpeI site close to the 5' end of the coding sequence allows for the fusion of the IL3 gene to any desired signal sequence with minimal requirement for further DNA synthesis.

CONSTRUCTION OF THE GENE The desired gene sequence was divided into 18 oligodeoxyribonucleotides (oligomers) as depicted in Figure 3. 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 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 BB407 and BB424 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 Figure 4. The ligation products were separated on a 2% low gelling temperature (LGT) gel and the band corresponding to the 429 bp IL3 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'1 ampicillin.Colonies containing potential clones were then grown up in Lbroth 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, 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 IL3 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, USA, 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, 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 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, 1mM EDTA, 0.18 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 158 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 -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 micro-filter.

The concentration of purified product was measured by determining its absorbance at 260 nm.

3) Rinsing 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 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 IL3 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 ? % 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 IL3 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'1 ampicillin.

9) Isolation of plasmid DNA Plasmid DNA was prepared from the colonies containing potential IL3 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 IL3 and having restriction sites for the following enzymes: HindIII, NcoI, SpI, HpaI, ClaI, Nsp(7524)V, NsiI, BalI, Eaqi, XmnI, EcoRI, BspMII, FspI, NheI, BamHI and EcoRI.

2. DNA including the following sequence: ATG GCT CCC ATG ACC CAG ACA ACT AGT TTG AAG ACA AGC TGG GTT AAC TGC TCT AAC ATG ATC GAT GAA ATT ATA ACA CAC TTA AAG CAG CCA CCT TTG CCT TTG CTG GAC TTC AAC AAC CTC AAT GGG GAA GAC CAA GAC ATT CTG ATG GAA AAT AAC CTT CGA AGG CCA AAC CTG GAG GCA TTC AAC AGG GCT GTC AAG AGT TTA CAG AAT GCA TCA GCA ATT GAG AGC ATT CTT AAA AAT CTC CTG CCA TGT CTG CCC CTG GCC ACG GCC GCA CCC ACG CGA CAT CCA ATC CAT ATC AAG GAC GGT GAC TGG AAT GAA TTC CGG AGG AAA CTG ACG TTC TAT CTG AAA ACC CTT GAG AAT GCG CAG GCT CAA CAG ACG ACT TTG AGG CTA GCG ATC TTT TAG

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.