Optimized nucleic acid sequences coding for the alpha-chain of human chorionic gonadotropin

Abstract

A host cell characterized in that it comprises integrated into its genome a sequence coding for the a chain of hCG, and use of the host cell to produce recombinant hCG.

Claims

1. A polynucleotide sequence comprising an optimized hCG -chain nucleic acid sequence selected from SEQ ID NO:1 and sequences having at least 97% sequence identity thereto, and SEQ ID NO:4 and sequences having at least 97% sequence identity thereto.

2. The polynucleotide sequence of claim 1, comprising the nucleic acid sequence of SEQ ID NO:1.

3. The polynucleotide sequence of claim 1, comprising a nucleic acid sequence having at least 97% sequence identity to SEQ ID NO:1.

4. The polynucleotide sequence of claim 1, comprising the nucleic acid sequence of SEQ ID NO:4.

5. The polynucleotide sequence of claim 1, comprising a nucleic acid sequence having at least 97% sequence identity to SEQ ID NO:4.

6. A cultured transformed cell having integrated into its genome an optimized hCG -chain polynucleotide sequence comprising SEQ ID NO:1 or a sequence having at least 96.5% sequence identity thereto or SEQ ID NO:4 or a sequence having at least 96.5% sequence identity thereto.

7. The transformed cell according to claim 6, wherein the integrated sequence comprises SEQ ID NO:1.

8. The transformed cell according to claim 6, wherein the integrated sequence comprises a sequence having at least 96.5% sequence identity to SEQ ID NO:1.

9. The transformed cell according to claim 6, wherein the integrated sequence comprises a sequence having at least 97% sequence identity to SEQ ID NO:1.

10. The transformed cell according to claim 6, wherein the integrated sequence comprises SEQ ID NO:4.

11. The transformed cell according to claim 6, wherein the integrated sequence comprises a sequence having at least 96.5% sequence identity to SEQ ID NO:4.

12. The transformed cell according to claim 6, wherein the integrated sequence comprises a sequence having at least 97% sequence identity to SEQ ID NO:4.

13. The transformed cell according to claim 6, wherein the transformed cell is the PER.C6 cell line deposited under ECACC No. 96022940 transformed to have integrated into its genome the optimized hCG chain polynucleotide sequence.

14. The transformed cell according to claim 6, further having integrated into its genome a cDNA encoding alpha-2,3-sialyltransferase.

15. The transformed cell according to claim 6, further having integrated into its genome a nucleic acid sequence encoding the hCG chain.

16. A method for producing recombinant protein in a cell, comprising culturing a cell according to claim 6 in a suitable medium, and harvesting the recombinant protein.

17. A method for producing recombinant hCG in a cell, comprising culturing a cell according to claim 13 in a suitable medium, and harvesting the recombinant protein.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) The present invention will now be described in more detail with reference to the following Examples and to the attached drawings in which:

(2) FIG. 1 shows the nucleic acid (SEQ ID NO: 1) and derived amino acid (SEQ ID NO: 6) sequences of the optimised hCG alpha sequence;

(3) FIG. 2 shows the nucleic acid (SEQ ID NO: 13) and derived amino acid sequences of hCG beta sequence (SEQ ID NO: 7);

(4) FIG. 3 shows the nucleic acid (SEQ ID NO: 14) and derived amino acid sequences of ST3GAL4 (SEQ ID NO: 8)

(5) FIG. 4 shows a plasmid map of the phCGalpha/beta expression vector;

(6) FIG. 5 shows the 2,3-sialyltransferase (ST3GAL4) expression vector;

(7) FIG. 6 shows the detection of rhCG Isoforms by IEF stained with Coomassie Blue in compositions according to the invention (Lanes 4, 5, 6 and 7, 15 g per lane); the CHO derived composition of the prior art, Ovitrelle (Lane 2, 15 g); and a human urinary product obtained from pregnant women Novarel (Lane 3, 15 g); and FIG. 7 compares the concentration of hCG (alpha-beta hCG) and free beta chain (beta hCG) produced using the cells of the invention including the Optimised nucleic sequence of the alpha sub unit, compared with concentration of hCG (alpha-beta hCG) and free beta chain (beta hCG) produced in comparative example cells including the WT (wild-type) alpha sub unit;

EXAMPLES 1 TO 7

(8) Overview of Cell Line Development Process

(9) A single plasmid carrying hCG alpha and beta chains, each driven from a separate CMVie promoter, was transfected into PER.C6 cells by electroporation.

(10) This plasmid carried a neomycin resistance gene cassette (FIG. 4) allowing for selection of transfectants using G418 (Geneticin). Pools of transfectants were expanded and then subjected to limiting dilution cloning in 96 well plates under G418 selection. Clonal lines were expanded and high expressing colonies were identified based on hCG titre in the cell culture supernatant.

(11) Five lead clones were selected, based on high productivity, and each was then transfected with a second plasmid expressing the 2,3-sialyltransferase gene ST3GAL4 along with the Hygromycin selection maker (FIG. 5). Each of the five transfection pools was expanded and cell culture supernatants were assessed for hCG concentration and in vivo pharmacokinetics (PK).

(12) Promising pools were then subjected to another round of limited dilution cloning. The resulting clones were expanded and cell culture supernatants were assessed for hCG concentration and exposed galactose on the hCG protein (by lectin ELISA). Those with a low level of exposed galactose were subjected to further assays including in vivo PK. Clones that produced hCG with optimum PK profiles, had low exposed galactose and expressed the protein at high levels were assessed and selected for productivity and growth characteristics.

Example 1

Sequence Selection and Plasmid Vectors

(13) The coding region of the gene for the hCG alpha polypeptide is shown in FIG. 1. The coding region of the gene for the hCG alpha polypeptide sequence is referred to herein SEQ ID NO: 1.

(14) The coding region of the gene for hCG beta polypeptide was used according to Fiddes and Goodman (1980). The sequence is banked as NP_000728 and is consistent with the protein sequences of CGbeta3, CGbeta5 and CGbeta7. The sequence is referred to herein as SEQ ID NO: 2

(15) The coding region of the gene for beta-galactoside alpha-2,3-sialyltransferase 4 (2,3-sialyltransferase, ST3GAL4) was used according to Kitagawa and Paulson (1994). The sequence is banked as L23767 and referred to herein as SEQ ID NO: 3.

(16) Two plasmids were used: the first, phCG, co-expresses hCG alpha and beta chains; and the second, expresses the sialyltransferase gene ST3GAL4.

Example 2a

Construction of the hCG Expression Vector

(17) phCG is a synthetic alpha chain of hCG, optimised for codon usage in mammalian cells including the native hCG alpha signal peptide. It may be engineered by methods well known in the art.

(18) The coding sequence of hCG alpha polypeptide (SEQ ID NO: 1) and hCG beta polypeptide (NP_000728, SEQ ID NO: 2) were amplified by PCR using methods well known in the art (see, for example, International Patent Application published as WO2011/042688). The phCG alpha DNA was digested with BamHI and NheI and inserted into the sites BamHI and NheI on a CMV driven mammalian expression vector (Crucell vector pcDNA3002Neo). This placed the gene in the correct orientation for expression driven by a CMVie promoter with a downstream BGH polyA signal. The native hCG beta gene including native signal peptide was digested with the restriction enzymes AscI and HpaI and inserted into the AscI and HpaI sites so that expression would be driven by a second CMVie promoter with an additional downstream BGH poly A signal. The vector backbone also included a neomycin resistance maker as well as elements required for selection and replication in prokaryotic cells. The vector was amplified and sequenced using general methods are known in the art. Sequences of the optimised hCG alpha and native (wild-type) beta chains are given in FIGS. 1 and 2, respectively.

(19) All colonies selected for sequencing contained the correct sequences according to SEQ ID NO: 1 and SEQ ID NO: 2. Plasmid phCG A+B was selected for transfection (FIG. 4).

Example 2b

Construction of the ST3 Expression Vector

(20) The ST3GAL4 gene is expressed in pST3. The coding sequence of beta-galactoside alpha-2,3-sialyltransferase 4 (ST3, L23767, SEQ ID NO: 3, FIG. 3) was amplified by PCR using the primer combination 2,3STfw and 2,3STrev.

(21) TABLE-US-00001 2,3STfw (SEQIDNO:11) 5'-CCAGGATCCGCCACCATGTGTCCTGCAGGCTGGAAGC-3' 2,3STrev (SEQIDNO:12) 5'-TTTTTTTCTTAAGTCAGAAGGACGTGAGGTTCTTG-3'

(22) The resulting amplified ST3 DNA was digested with the restriction enzymes BamHI and AflII and inserted into the BamHI and AflII sites on the CMV driven mammalian expression vector carrying a hygromycin resistance marker (vector pcDNA3002Neo) so that it is located downstream of CMVie promoter and upstream of BGH polyA sequence. The vector backbone also included a hygromycin resistance maker as well as elements required for selection and replication in prokaryotic cells. The vector was amplified as previously described and sequenced. Clone pST3#1 (FIG. 5) contained the correct sequence according to SEQ ID NO: 3 and was selected for transfection.

Example 3

Plasmid Transfection

(23) The plasmid phCG (FIG. 4), which has a single PvuI site located in the prokaryotic beta-lactamase gene, was linearised with PvuI (New England Biolabs Cat. No. R0150). Linearized plasmid DNA was transfected into PER.C6 cells as follows.

(24) Cell cultures were maintained in complete PERMAB medium (CD4PERMAB (Hyclone Cat. No. SH30871.01) supplemented with L-glutamine to 3 mM final concentration (Invitrogen Cat. No. 25030-123) and Pluronic F68 at 1.0 g/L final concentration (Invitrogen Cat. No. 24040-032) in 250 ml Erlenmeyer flasks. Cells were maintained in a shaking incubator (Kuhner Climo-shaker ISF1-X) set at 100 rpm, 5% CO.sub.2 and 37 C., for at least 14 days prior to transfection. 48 hours prior to transfection, cells were transferred into fresh medium at a density of 0.510.sup.6 cells/ml.

(25) On the day of transfection, cells were counted in a Beckman Coulter ViCell XR to determine cell density and to ensure viability was >90%. Cells were harvested by centrifugation and resuspended in fresh PERMAB medium before being mixed with linearized phCG DNA. The cell/DNA mix was electro-shocked in the chamber of an electroporator set at 250V for 5 msec, before being quickly transferred to 10 ml of pre-warmed PERMAB medium. This process was repeated a total of 6 times and all 6 transfections were pooled into a single T-175 cm.sup.2 tissue culture flask. The flask was placed in a static incubator set at 37 C., 5% CO.sub.2. After 48 hours, cells were resuspended in the appropriate volume of selective PERMAB (complete PERMAB medium+G418 (125 g/ml)) to give a viable cell density of 0.510.sup.6/ml.

(26) The pool culture was passaged twice weekly, maintaining cells at a density of 0.310.sup.6 cell/ml in selective medium until cell viability had increased to >50%. At this point, cells were transferred to shaking cultures in 250 ml Erlenmeyer flasks. After several weeks in shaking culture, pool supernatant was sampled and assayed for hCG concentration. Once it was established that the pool was positive for hCG expression, cells were prepared for limited dilution cloning.

(27) A cell suspension at 0.3 viable cells/ml was prepared in PERMAB medium supplemented with G-418 at 125 g/ml. The cell suspension was dispensed into 96 well flat bottomed tissue culture plates at 200 l/well and incubated in a humidified atmosphere at 37 C., 5% CO.sub.2 (Binder CB150). Plates were scanned regularly using the Genetix Clone Select Imager to track the growth of cells in each well.

(28) After two weeks, 535 wells were identified that contained actively growing colonies of cells. Supernatants from these wells were sampled and assayed for hCG using a commercial kit (DRG diagnostics HCG ELISA Cat. No. EIA1469). Based on the results from these assays, a total of 162 of the colonies were transferred into 24 well plates containing 0.5 ml/well selective PERMAB medium. When cells in the wells were near to confluency, supernatant from each of the 162 wells was sampled and assayed for hCG levels. Based on these results, 91 of the best expressing cell lines were selected for expansion into T-25 flasks. These cell lines were again grown to near confluence, at which point supernatants were sampled and assayed for hCG as above. Based on these results, the 58 best expressing cell lines were expanded into T-75 flasks. Specific production rate (SPR) analysis was performed on each of these 58 cell lines by methods known in the art and specific productivity was expressed in pg/cell/day.

(29) Thus, in this Example a plasmid incorporating hCG alpha and beta chains was transfected into PER.C6 cells and transfectants were selected using medium containing G418. Growing colonies of cells were screened for hCG concentration in the supernatant and those expressing the highest level were expanded further. After subsequent rounds of expansion and screening, 20 clones that expressed hCG were selected for growth and productivity studies. Based on the results of these studies, 5 clones were selected for transfection with a second plasmid incorporating the ST3GAL4 gene.

Example 4

Transfection with the ST3GAL4 Plasmid pST3

(30) Stable clones were generated as previously described in Example 3.

(31) The five best clones produced by the method as described in Example 3 were selected for transfection with the ST3GAL4 plasmid pST3 (see Example 2, FIGS. 3 and 5). Transfection was performed by electroporation using the same method described in Example 3, except that transformants were selected in complete PERMAB medium supplemented with Hygromycin at 0.5 g/ml instead of G418. Using this method, five pools were obtained that expressed hCG.

(32) Samples of supernatants from the transfection pools were assessed in a rat pharmacokinetic model and, based on this data along with data from the RCA-lectin binding assay (measuring exposed galactose on the hCG chains), a single pool was selected for further limited dilution cloning, by methods known in the art.

(33) This dilution cloning yielded >600 clones each of which was also screened for hCG expression and the degree of exposed galactose as measured by RCA-lectin binding. Those clones with the lowest levels of exposed galactose were expanded further and samples were subjected to in vivo PK and corroborating lectin binding data. Five clones derived from the single pool were further assessed for growth and productivity characteristics.

(34) Each of these clones was expanded and a seed stock cell bank was made following standard cryopreservation procedures. Stocks from the seed stock cell bank were thawed and found to be viable.

Example 5

Analysis by Isoelectric Focussing

(35) Electrophoresis is defined as the transport of charged molecules through a solvent by an electrical field. The mobility of a biological molecule through an electric field will depend on the field strength, net charge on the molecule, size and shape of the molecule, ionic strength and properties of the medium through which the molecules migrate.

(36) Isoelectric focusing (IEF) is an electrophoretic technique for the separation of proteins based on their pI. The pI is the pH at which a protein has no net charge and will not migrate in an electric field. The sialic acid content of the hCG isoforms subtly alters the pI point for each isoform, which can be exploited using this technique to visualise the Per.C6 hCG isoforms from each clone.

(37) The isoelectric points of the Per.C6 produced hCG isoforms were analyzed using isoelectric focussing. Per.C6 hCG was produced as described in Example 6.

(38) Per.C6 hCG samples were separated on Novex IEF Gels containing 5% polyacrylamide under native conditions on a pH 3.0-7.0 gradient in an ampholyte solution pH 3.0-7.0. Proteins were visualised using Coomassie Blue staining, using methods well known in the art.

(39) FIG. 6 shows the detection of rhCG Isoforms by IEF stained with Coomassie Blue in compositions produced according to the invention produced from the cloned cell lines made by the method set out in Examples 6 (Lanes 4, 5, 6 and 7, 15 g per lane); the CHO derived composition of the prior art, Ovitrelle (Lane 2, 15 g); and a human urinary product obtained from pregnant women Novarel (Lane 3, 15 g). The bands represent isoforms of hCG containing different numbers of sialic acid molecules. FIG. 6 indicates that human cell line derived recombinant hCG engineered with 2,3-sialyltransferase (compositions according to the invention) is more acidic than Ovitrelle and urinary hCG from pregnant women.

(40) FIG. 7 compares the concentration of hCG (alpha-beta hCG) and free beta chain (beta hCG) produced using the cells of the invention (that is, using cell lines made by the method set out in Examples 1 to 4) which include the Optimised alpha sub unit, compared with concentration of hCG (alpha-beta hCG) and free beta chain (beta hCG) produced in comparative example cells including the WT (wild-type) alpha sub unit. FIG. 7 shows that the cells of the invention express high amounts of hCG and low excess of the free beta subunit. In other words, the yield and purity of recombinant hCG produced by the cells and methods of the invention is markedly improved.

(41) Table A indicates the percentage of free -hCG Subunits in semi-purified samples of various batches of hCG produced using the cells of the invention (that is, using cell lines made by the method set out in Examples 1 to 4) which include the Optimised alpha sub unit (Opt. alpha hCG), compared with the percentage of free -hCG Subunits in semi-purified samples of hCG produced in comparative example cells including the WT (wild-type) alpha sub unit (WT alpha hCG), as determined by Hydrophobic Phenyl-5PW HPLC chromatography.

(42) Table A shows that the cells of the invention express high amounts of hCG and a rather lower excess of the free beta subunit (5.7-10.6%) compared to cells including the WT alpha sub unit (64-66%). In other words, the yield and purity of recombinant hCG produced by the cells and methods of the invention is markedly improved.

(43) TABLE-US-00002 TABLE A % Free -hCG Clone (PHE-5PW) WT alpha hCG (1G2) 64.1 WT alpha hCG (1G2) 66.0 Opt. alpha hCG 5.7 (13.8) Opt. alpha hCG (29) 7.5 Opt. alpha hCG (29) 6.6 Opt. alpha hCG (29) 10.6

Example 6

Production and Purification Overview

(44) A procedure was developed to produce recombinant hCG in PER.C6 cells that were cultured in suspension in serum free medium. The procedure is described below and was applied to several hCG-producing PER.C6 cell lines.

(45) Recombinant hCG from an 2,3-sialyltransferase transfected clone was prepared using the methods of Examples 1 to 4 described above.

(46) The cells were grown in shaker flasks in 6GRO medium (SAFC) until a cell density of 110.sup.6 to 310.sup.6 cells/ml was achieved. The cells were transferred to a 5 L glass stirred tank bioreactor with a density of about 110.sup.6 cells/ml. The bioreactor worked in perfusion mode using a Proper1 media (Lonza).

(47) Thereafter, purification of the product rhCG was carried out using various ultrafiltration steps, anion and cation exchange capture chromatography, hydrophobic chromatography and pseudo-affinity chromatography, by methods well known in the art.

(48) During all chromatographic procedures, the presence of immunoreactive recombinant hCG was confirmed by ELISA (DRG EIA 1469) and IEF (Example 5).

Example 7

Sialic Acid Content

(49) Sialic acid is a protein-bound carbohydrate considered to be a mono-saccharide and occurs in combination with other mono-saccharides like galactose, mannose, glucosamine, galactosamine and fucose. The total sialic acid on purified rhCG according to the invention was measured using a method based on the method of Stanton et. al. (J. Biochem. Biophys. Methods. 30 (1995), 37-48).

(50) The total sialic acid content of samples of Per.C6 recombinant hCG modified with 2,3-sialyltransferase (produced by the methods of Example 4 and 6) were measured and the results are in Table 1 below [expressed in terms of a ratio of moles of sialic acid to moles of protein].

Example 8

hCG Bioassay According to USP

(51) A hCG Bioassay was carried out, in order to determine the hCG specific activity, for each of the samples of Table 1 below. The activity was measured according to USP (USP Monographs: Chorionic Gonadotropin, USPC Official Aug. 1, 2009 -Nov. 30, 2009), using Ovitrelle as a standard. Ovitrelle has a biological activity of 26,000 IU/mg (Curr Med Res Opin. 2005 December; 21(12): 1969-76). The acceptance limit was >21,000 IU hCG/mg. The biological activity for samples of human cell line derived hCG recombinant hCGs engineered with 2,3-sialyltransferase are shown in Table 1.

(52) TABLE-US-00003 TABLE 1 Sample 1 2 3 4 5 6 Sialic acid 13 13 13 13 13 15 (SA) content (mol SA/ molhCG) Potency IU/mg 25363 25009 24904 24623 25645 26623 % Potency 97.5 96.2 95.8 94.7 98.6 102.4 (Ovitrelle 26000 IU/mg) Sample 7 8 9 10 11 Sialic acid 14 14 13 13 13 (SA) content (mol SA/ molhCG) Potency IU/mg 27227 26142 26539 26181 24230 % Potency 104.7 100.5 102 101 93.2 (Ovitrelle 26000 IU/mg)

(53) As seen above, the potency is similar to, and may be greater than, Ovitrelle (see e.g. Sample 7).

REFERENCES

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(55) TABLE-US-00004 OptimisedhCGalpha NucleotidesequenceofoptimisedhCGalpha (SEQIDNO:1) 1ATGGACTACTACCGGAAGTACGCCGCCATCTTCCTGGTGACCCTGAGCGTGTTCCTGCAC 61GTGCTGCACAGCGCCCCTGACGTGCAGGACTGCCCCGAGTGCACCCTGCAGGAAAACCCC 121TTCTTCAGCCAGCCTGGCGCCCCTATCCTGCAGTGCATGGGCTGCTGCTTCAGCAGAGCC 181TACCCCACCCCCCTGCGGAGCAAGAAAACCATGCTGGTGCAGAAAAACGTGACCAGCGAG 241AGCACCTGCTGCGTGGCCAAGAGCTACAACCGGGTGACCGTGATGGGCGGCTTCAAGGTG 301GAGAACCACACCGCCTGCCACTGCAGCACCTGCTACTACCACAAGTCCT ProteinsequenceofhCGoptimisedalpha(SEQIDNO:6) 1MDYYRKYAAIFLVTLSVFLHVLHSAPDVQDCPECTLQENPFFSQPGAPILQCMGCCFSRA 61YPTPLRSKKTMLVQKNVTSESTCCVAKSYNRVTVMGGFKVENHTACHCSTCYYHKS HumanChorionicGonadotrophinbetapolypeptide AccessionnumberNP_000728 NucleotidesequenceofhCGbeta (SEQIDNO:2) Nucleotidesequence 1ATGGAGATGTTCCAGGGGCTGCTGCTGTTGCTGCTGCTGAGCATGGGCGGGACATGGGCA 61TCCAAGGAGCCGCTTCGGCCACGGTGCCGCCCCATCAATGCCACCCTGGCTGTGGAGAAG 121GAGGGCTGCCCCGTGTGCATCACCGTCAACACCACCATCTGTGCCGGCTACTGCCCCACC 181ATGACCCGCGTGCTGCAGGGGGTCCTGCCGGCCCTGCCTCAGGTGGTGTGCAACTACCGC 241GATGTGCGCTTCGAGTCCATCCGGCTCCCTGGCTGCCCGCGCGGCGTGAACCCCGTGGTC 301TCCTACGCCGTGGCTCTCAGCTGTCAATGTGCACTCTGCCGCCGCAGCACCACTGACTGC 361GGGGGTCCCAAGGACCACCCCTTGACCTGTGATGACCCCCGCTTCCAGGACTCCTCTTCC 421TCAAAGGCCCCTCCCCCCAGCCTTCCAAGTCCATCCCGACTCCCGGGGCCCTCGGACACC 481CCGATCCTCCCACAATAA ProteinsequenceofhCGbeta(SEQIDNO:7) 1MEMFQGLLLLLLLSMGGTWASKEPLRPRCRPINATLAVEKEGCPVCITVNTTICAGYCPT 61MTRVLQGVLPALPQVVCNYRDVRFESIRLPGCPRGVNPVVSYAVALSCQCALCRRSTTDC 121GGPKDHPLTCDDPRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ Beta-galactosidealpha-2,3-sialyltransferase4 AccessionNumberL23767 NucleotidesequenceofST3GAL4 (SEQIDNO:3) 1ATGTGTCCTGCAGGCTGGAAGCTCCTGGCCATGTTGGCTCTGGTCCTGGTCGTCATGGTG 61TGGTATTCCATCTCCCGGGAAGACAGGTACATCGAGCTTTTTTATTTTCCCATCCCAGAG 121AAGAAGGAGCCGTGCCTCCAGGGTGAGGCAGAGAGCAAGGCCTCTAAGCTCTTTGGCAAC 181TACTCCCGGGATCAGCCCATCTTCCTGCGGCTTGAGGATTATTTCTGGGTCAAGACGCCA 241TCTGCTTACGAGCTGCCCTATGGGACCAAGGGGAGTGAGGATCTGCTCCTCCGGGTGCTA 301GCCATCACCAGCTCCTCCATCCCCAAGAACATCCAGAGCCTCAGGTGCCGCCGCTGTGTG 361GTCGTGGGGAACGGGCACCGGCTGCGGAACAGCTCACTGGGAGATGCCATCAACAAGTAC 421GATGTGGTCATCAGATTGAACAATGCCCCAGTGGCTGGCTATGAGGGTGACGTGGGCTCC 481AAGACCACCATGCGTCTCTTCTACCCTGAATCTGCCCACTTCGACCCCAAAGTAGAAAAC 541AACCCAGACACACTCCTCGTCCTGGTAGCTTTCAAGGCAATGGACTTCCACTGGATTGAG 601ACCATCCTGAGTGATAAGAAGCGGGTGCGAAAGGGTTTCTGGAAACAGCCTCCCCTCATC 661TGGGATGTCAATCCTAAACAGATTCGGATTCTCAACCCCTTCTTCATGGAGATTGCAGCT 721GACAAACTGCTGAGCCTGCCAATGCAACAGCCACGGAAGATTAAGCAGAAGCCCACCACG 781GGCCTGTTGGCCATCACGCTGGCCCTCCACCTCTGTGACTTGGTGCACATTGCCGGCTTT 841GGCTACCCAGACGCCTACAACAAGAAGCAGACCATTCACTACTATGAGCAGATCACGCTC 901AAGTCCATGGCGGGGTCAGGCCATAATGTCTCCCAAGAGGCCCTGGCCATTAAGCGGATG 961CTGGAGATGGGAGCTATCAAGAACCTCACGTCCTTCTGA ProteinSequenceofST3GAL4(SEQIDNO:8) 1MCPAGWKLLAMLALVLVVMVWYSISREDRYIELFYFPIPEKKEPCLQGEAESKASKLFGN 61YSRDQPIFLRLEDYFWVKTPSAYELPYGTKGSEDLLLRVLAITSSSIPKNIQSLRCRRCV 121VVGNGHRLRNSSLGDAINKYDVVIRLNNAPVAGYEGDVGSKTTMRLFYPESAHFDPKVEN 181NPDTLLVLVAFKAMDFHWIETILSDKKRVRKGFWKQPPLIWDVNPKQIRILNPFFMEIAA 241DKLLSLPMQQPRKIKQKPTTGLLAITLALHLCDLVHIAGFGYPDAYNKKQTIHYYEQITL 301KSMAGSGHNVSQEALAIKRMLEMGAIKNLTSF OptimisedhCGalphachain NucleotidesequenceofoptimisedhCGalphachain (SEQIDNO:4) 1GCCCCTGACGTGCAGGACTGCCCCGAGTGCACCCTGCAGGAAAACCCCTTCTTCAGCCAG 61CCTGGCGCCCCTATCCTGCAGTGCATGGGCTGCTGCTTCAGCAGAGCCTACCCCACCCCC 121CTGCGGAGCAAGAAAACCATGCTGGTGCAGAAAAACGTGACCAGCGAGAGCACCTGCTGC 181GTGGCCAAGAGCTACAACCGGGTGACCGTGATGGGCGGCTTCAAGGTGGAGAACCACACC 241GCCTGCCACTGCAGCACCTGCTACTACCACAAGTCCT ProteinsequenceofhCGoptimisedalphachain(SEQIDNO:9) 1APDVQDCPECTLQENPFFSQPGAPILQCMGCCFSRAYPTPLRSKKTMLVQKNVTSESTCC 61VAKSYNRVTVMGGFKVENHTACHCSTCYYHKS hCGalphapolypeptide AccessionnumberAH007338 NucleotidesequenceofhCGalpha (SEQIDNO:5) 1ATGGATTACTACAGAAAATATGCAGCTATCTTTCTGGTCACATTGTCGGTGTTTCTGCAT 61GTTCTCCATTCCGCTCCTGATGTGCAGGATTGCCCAGAATGCACGCTACAGGAAAACCCA 121TTCTTCTCCCAGCCGGGTGCCCCAATACTTCAGTGCATGGGCTGCTGCTTCTCTAGAGCA 181TATCCCACTCCACTAAGGTCCAAGAAGACGATGTTGGTCCAAAAGAACGTCACCTCAGAG 241TCCACTTGCTGTGTAGCTAAATCATATAACAGGGTCACAGTAATGGGGGGTTTCAAAGTG 301GAGAACCACACGGCGTGCCACTGCAGTACTTGTTATTATCACAAATCTTAA ProteinsequenceofhCGalpha(SEQIDNO:10) 1MDYYRKYAAIFLVTLSVFLHVLHSAPDVQDCPECTLQENPFFSQPGAPILQCMGCCFSRA 61YPTPLRSKKTMLVQKNVTSESTCCVAKSYNRVTVMGGFKVENHTACHCSTCYYHKS