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|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 the production of thyroid stimulating hormone comprising the steps of:
a) providing a first vector comprising a promoter, a DNA fragment encoding for the alpha subunit of said thyroid stimulating hormone and comprising at least one intron, and a terminating sequence, and a second vector comprising a promoter, a DNA fragment TSHB 1.2 or TSHB 2.0 encoding for the beta subunit of said thyroid stimulating hormone consisting essentially of coding exons II and III separated by an endogenous intervening sequence, and one intron about 300 base pairs in length positioned upstream of and adjacent to exon II, and a terminating sequence;
b) transforming host cells with said vectors; and
c) culturing said transformed cells under conditions whereby said thyroid stimulating hormone is produced.
2. The method of claim 1 wherein said DNA fragment encoding said .alpha. subunit further comprises a plurality of introns, 3' and 5' flanking regions, endogenous 5' untranslated sequence and polyadenylation signal.
3. The method of claim 1 wherein said selected dimeric protein is human thyroid stimulating hormone.
4. The method of claim 1 wherein said selected dimeric protein is non-human thyroid stimulating hormone.
5. The method of claim 3 wherein the DNA fragment for the beta subunit of human thyroid stimulating hormone is a 1.2 kb DNA fragment containing the two coding exons separated by an endogenous intervening sequence, but without the endogenous polyadenylation signal or additional 3' flanking sequence.
6. The method of claim 3 wherein the DNA fragment for the beta subunit of human thyroid stimulating hormone is a 2.0 kb DNA fragment containing the two coding exons separated by an endogenous intervening sequence, and including the endogenous polyadenylation signal and 0.8 kb of additional 3' flanking sequence.
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