Generated: April 25, 2017
|Title:||Heteropolymeric protein production methods|
|Abstract:||Cultured mammalian cells 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)|
1. A method for accumulating, in a host cell, mRNA encoding the .alpha.-subunit of a dimeric hormone selected from the group consisting of luteinizing hormone, follicle
stimulating hormone, chorionic gonadotropin and thyroid stimulating hormone, comprising:
transforming the host cell with a vector comprising a promoter, a structural gene encoding the .alpha.-subunit, and a terminating sequence being operatively linked to permit expression of said gene by the host cell, 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 the structural gene encoding the .alpha.-subunit, is not the entire genomic sequence of the gene encoding the .alpha.-subunit of bovine luteinizing hormone; and
culturing said transformed cells under conditions by which mRNA encoding said .alpha.-subunit is produced,
wherein said intron immediately 5' to the coding region of the .alpha.-subunit is spatially disposed with respect to the ATG of the coding region such that an amount of mRNA encoding said .alpha.-subunit 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 includes two introns.
3. A method in accordance with claim 1 wherein said structural gene is a cDNA encoding the .alpha.-subunit.
4. A method in accordance with claim 1 wherein said intron immediately 5' to the coding region is a homologous intron.
5. A method in accordance with claim 1 wherein said intron immediately 5' to the coding region is a heterologous intron.
6. A method in accordance with claim 1, wherein said structural gene encoding the .alpha.-subunit is not the entire genomic sequence of the gene encoding said .alpha.-subunit.
7. A method in accordance with claim 1, wherein, in the structural gene encoding the .alpha.-subunit, said one intron which is immediately 5' to the coding region of the .alpha.-subunit naturally occurs at that position in the genomic sequence of the gene encoding the .alpha.-subunit of the natural dimeric protein corresponding to the dimeric protein being produced.
8. A method in accordance with claim 1, wherein said structural gene contains only a single intron.
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