GB2217327A - Synthetic gene encoding interleukin-6 - Google Patents

Abstract

Synthetic DNA coding for human interleukin 6 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 interleukin 6.

Human interleukin 6 (IL6) is the name given to a factor that has been variously described as Interferon b2, hybridoma growth factor and as a B cell differentiation factor (BSFII) that induces the final maturation of B cells into antibody secreting plasma cells.

Purification of BSFII from a T cell line that produced this activity in substantial amounts allowed partial sequence determination to be obtained which in turn allowed the design of probes and the isolation of cDNA clones. One clone was shown to direct the synthesis of an identical activity when expressed transiently in COS cells.

Sequence analysis of the cDNA clone has revealed that it encodes a polypeptide of 212 amino acids and allowed its identity with the other factors mentioned above to be established. The deduced protein sequence has a stretch of hydrophobic amino acids at the N terminus that is characteristic of a secretion signal. The predicted cleavage site at which processing would occur on export is after residue 28 which would yield a mature protein of 184 amino acids. The sequence contains two potential N glycosylation sites.

The various activities now attributed to IL6 have all been described previously: IFNb2 (Weissenbach et al.

P.N.A.S 77, 7152 (1980)), HGF (Astaldi et al. J.

Immunol. 125, 144 (1980)) and BSFII (Hirano et al.

P.N.A.S. 82, 5490 (1985)). The sequence of BSFII has been described (Hirano et al. Nature 324, 73 (1986)).

This paper and those of Haegeman et al. (Eur. J.

Biochem 159, 625 (1986)) and Zilberstein et al. (EMBO Journal 5, 2529 (1986)) clearly establish that all three activities are vested in the same protein.

The construction of a synthetic gene encoding human IL6 is not described in the above documents.

In order to facilitate the dissection of the structure/function relationships of human IL6, its incorporation into expression vectors and the production of novel chimaeric proteins containing IL6 functionality an improved novel synthetic gene for human IL6.

It is by no means easy to predict the design of an improved IL6 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 synthetic IL6 genes which are distinct from the natural IL6 gene and have advantages in the ease with which they can be modified due to the presence of useful restriction sites.

According to a first aspect of the invention, there is provided DNA coding for human IL6 and having restriction sites for the following enzymes: Hin DIII, B@pMI, BstXI, EcoRV, NheI, BspMII, XhoI, SalI, AlwNI, PstI, XbaI, NsiI, PstI, AlwNI, PstI, FspI, PstI, MstII, BamHI and EcoRI According to a second aspect of the invention, there is provided DNA including the following sequence:: A AGC TTA CCT GCC ATG CCA GTA CCC CCA GGA GAA GAT TCC AAA GAT GTG GCC GCC CCA CAC AGA CAG CCA CTC ACC TCT TCA GAA CGA ATT GAC AAA CAA ATT CGG TAC ATC CTC GAC GGG ATA TCA GCC CTG AGA AAG GAG ACA TGT AAC AAG AGT AAC ATG TGT GAA AGC AGC AAA GAG GCG CTA GCA GAA AAC AAC CTG AAC CTT CCA AAG ATG GCT GAA AAA GAT GGA TGC TTC CAA TCC GGA TTC AAT GAG GAG ACT TGC CTG GTG AAA ATC ATC ACT GGT CTT TTG GAG TTT GAG GTA TAC CTC GAG TAC CTC CAG AAC AGA TTT GAG AGT AGT GAG GAA CAA GCC AGA GCT GTG CAG ATG TCG ACA AAA GTC CTG ATC CAG TTC CTG CAG AAA AAG GCA AAG AAT CTA GAT GCA ATA ACC ACC CCT GAC CCA ACC ACA AAT GCA TCC CTG CTG ACG AAG CTG CAG GCA CAG AAC CAG TGG CTG CAG GAC ATG ACA ACT CAT CTC ATT CTG CGC AGC TTT AAG GAG TTC CTG CAG TCC AGC CTG AGG GCT CTT CGG CAA ATG TAG TAA GGA TCC GAA TTC The design for the synthetic mature human IL6 gene was based on the published cDNA sequence (Figure 1) 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. It was not possible to engineer an NcoI site to encompass the ATG initiator codon as the first codon of the mature IL6 gene commences with a C residue though a favourable translation initiation sequence was provided based on the rules defined by Kozak (Kozak, N. Cell 44, 283-292 (1986)). A BstXI site close to the 5' end of the coding sequence allows for the fusion of the IL6 gene to any desired signal sequence with minimal requirement for further DNA synthesis.

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 nonpalindromic sequence of six base pairs (5'-ACCTGC-3') and gives rise to a staggered cut in the DNA four bases in downstream of the recognition site resulting 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 Sl or Mung Bean nuclease treatment will result in a blunt end following the ATG. This approach cannot be used, however, when the codon following the ATG commences with other than a G residue (as is the case with the IL6 gene) . 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.

The synthetic gene is designed primarily for expression in higher eukaryotic systems, particularly mammalian cells but we would expect it 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 cDNA sequence for human IL6 together with the deduced amino acid sequence; Figure 2 illustrates the utility of a BsmMI site prior to the structural gene; Figure 3 shows the sequence of a synthetic gene for human IL6 along with location of useful restriction sites; Figure 4 shows the sequence of the IL6 synthetic gene of Figure 3 divided into oligonucleotides; and Figure 5 shows a summary of the assembly procedure used.

CONSTRUCTION OF THE GENE The desired gene sequence was divided into 28 oligodeoxyribonucleotides (oligomers) as depicted in Figure 4. The division was couch as to provide 7 base cohesive ends after annealing complementary pairs of oligomers with the exception of the cohesive end between oligomers BB556/BB557 and BB558/BB559 which is 5 bases long. 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 phophoramidite 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 BB552 and BB579 were then kinased to provide them with a 5' phosphate as required for the ligation step. Complementary oligomers were then annealed and the 14 pairs of oligomers ligated together with 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 580/580 bp IL6 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 -l ampicillin. Colonies containing potential clones were then grown up in Lbroth containing ampicillin at 100 mcg.m1 -l 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 IL6 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, 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.

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 OPG 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 oligome 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, l0mM tris, 10 mM borate, lmM 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 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 % ethano', 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) 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 Gel2, 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 IL6 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 90 LGT gel and the vector band purified as described above.

20 ng of cut vector DNA was then ligated to various quantities of IL6 DNA ranging from 2 to 20 ng for 4 hr using the ligation buffer described above. The ligation products were used to transform competent How87 as has been described. Ampicillin resistant transformants were selected on L-agar plates containing 100 mcg.ml-l ampicillin.

9) Isolation of plasmid DNA Plasmid DNA was prepared from the colonies containing potential IL6 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 all salK hsdR rPsL srl recA56 Any other standard cloning recipient such as HB101 would be adequate.

Claims (11)

1. DNA coding for human IL6 and having restriction sites for the following enzymes: HinDIII, BsMI, BstXI, EcoRV, NheI, BspMII, XhoI, SalI, AlwNI, PstI, XbaI, NsiI, PstI, AlwNI, PstI, FspI, PstI, MstII, BamHI and EcoRI

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

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 3.