Plasmids, their preparation and their use in the manufacture...

Details Plasmids, their preparation and their use in the manufacture... Plasmids, their preparation and their use in the manufacture...

C - Chemistry, Metallurgy

12

N

195/1.2, 195/1.2

C12N 15/71 (2006.01) C12N 9/68 (2006.01) C12N 9/72 (2006.01) C12N 15/67 (2006.01) C12N 15/70 (2006.01) C12P 21/02 (2006.01)

Patent

CA 2020656

Plasmids containing synthetic DNA sequences are described which are suitable for the expression of the intermediate protein of the rscu-PA (i.e. the unglycosylated protein moiety of the single chain prourokinase) in Enterobacteriaceae, especially in E.coli, with expression rates far higher than those obtainable according to the state of the art. The plasmids according to the invention are characterized in that their operons include a regulatable, optionally synthetic promotor, a Shine-Dalgarno sequence, a start codon, a synthetic structural gene and downstream of the structural gene 1 to 2 terminators. The preparation of the plasmids as well as that of the synthetic DNA-sequences starting from commercially available plasmids etc. is described. The intermediate protein expressed in high yields by using the plasmids for transforming suitable hosts may be refolded in a manner known per se to the therapeutically (as plasminogen activator) useful rscu-PA which also optionally may serve as a starting material for obtaining the two chain unglycosylated urokinase protein (rtcu-PA) on a technical scale by splitting the single chain product in a manner known per se.

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