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Claims for Patent: 5,602,006

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Claims for Patent: 5,602,006

Title: Heteropolymeric protein production methods
Abstract:Cultured mammalian cell transfected with new vectors comprising full-length or partial .alpha. and .beta. subunit genomic DNA sequences produce significantly higher levels of dimeric glycoprotein hormone than do cells transfected with .alpha. and .beta. subunit cDNA sequences. In cases where only the cDNA clones are available, the cDNA sequences can be used in new expression vectors comprising introns or other important genomic regions from a homologous or heterologous source.
Inventor(s): Kelton; Christie A. (Hopkinton, MA), Nugent; Noreen P. (Framingham, MA), Chappel; Scott C. (Boston, MA)
Assignee: Genzyme Corporation (Cambridge, MA)
Application Number:08/455,396
Patent Claims: 1. A method for increasing the production of a protein in a host cell transformed with a vector comprising DNA encoding for said protein, wherein said protein is the .alpha.-subunit of a dimeric protein selected from the group consisting of luteinizing hormone, follicle stimulating hormone, chorionic gonadotropin and thyroid stimulating hormone, comprising:

(a) providing a vector comprising a promoter, a structural gene encoding said .alpha.-subunit of said dimeric protein and a terminating sequence, said promoter, structural gene and terminating sequence being operatively linked to permit expression of said gene when a host cell is appropriately transformed by said vector, wherein said structural gene comprises a coding region and at least one intron, wherein one intron is immediately 5' to the coding region of the .alpha.-subunit, with the proviso that said structural gene is not the entire genomic sequence of the gene encoding said .alpha.-subunit;

(b) transforming host cells with said vector; and

(c) culturing said transformed cells under conditions by which the .alpha.-subunit protein is produced,

whereby an amount of .alpha.-subunit protein is produced which is greater than that which can be produced under comparable conditions using a structural gene encoding the .alpha.-subunit which is the cDNA without introns.

2. A method in accordance with claim 1, wherein said structural gene comprises DNA encoding the amino acid sequence Met Asp Tyr Tyr Arg Lys Tyr Ala Ala Ile Phe Leu Val Thr Leu Ser Val Phe Leu His Val Leu His Ser Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Ash Pro Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys Thr Met Leu Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser.

3. A method in accordance with claim 1, wherein said structural gene contains only a single intron.

4. A method in accordance with claim 1, wherein said structural gene is produced by adding, by recombinant DNA techniques, said at least one intron to the cDNA encoding said .alpha.-subunit.

5. A method in accordance with claim 1, wherein said one intron which is immediately 5' to the coding region of the .alpha.-subunit naturally occurs in the genomic sequence of the gene encoding the .alpha.-subunit of the natural dimeric protein corresponding to the dimeric protein the .alpha.-subunit of which is being produced.

6. A method in accordance with claim 5, wherein said structural gene comprises Met Asp Tyr Tyr Arg Lys Tyr Ala Ala Ile Phe Leu Val Thr Leu Ser Val Phe Leu His Val Leu His Ser Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys Thr Met Leu Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser.

7. A method in accordance with claim 5, wherein said structural gene contains only a single intron.

8. A method in accordance with claim 5, wherein said structural gene is produced by adding, by recombinant DNA techniques, said at least one intron to the cDNA encoding said .alpha.-subunit.
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