Plasmids, their construction and their use in the manufacture of

Details Plasmids, their construction and their use in the manufacture of Plasmids, their construction and their use in the manufacture of

1990-07-12

1997-06-10

Fleisher, Mindy

Chemistry: molecular biology and microbiology

Vector, per se

435 691, 435215, 43525233, 435 696, 435 711, 536 231, C12N 1500, C12P 2106, C07H 2102

Patent

active

056375034

ABSTRACT:
Plasmids containing synthetic DNA sequences are described which are suitable for the expression of the intermediate protein of the recombinant scu-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 prior methods. The plasmids comprise operons that include a regulatable, optionally synthetic, promotor, a Shine-Dalgarno sequence, a start codon, a synthetic structural gene with selected codon usage, and downstream of the structural gene, 1 to 2 transcription terminators. The preparation of the plasmids and of synthetic DNA-sequences starting from commercially available plasmids and intermediates is described. The intermediate protein, expressed in high yields by using the plasmids for transforming suitable hosts, may be refolded to produce the therapeutically useful plasminogen activator recombinant scu-PA. This protein also may serve as a starting material for obtaining the recombinant two chain unglycosylated urokinase protein (rtcu-PA) on a large scale by splitting the single chain product.

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