E. coli clone producing human pre-pro-urokinase - useful as thrombolytic agent

摘要

Prodn. of an E.coli clone which produces human preprourokinase (I) comprises first extracting and purifying total messenger RNA from human urokinase (II)-producing cells, and using the prod. for in vitro synthesis of complementary ds-DNA. - This is enriched in components at least large enough to code for (II), the prod. giving cohesive ends and inserted into a plasmid vector having complementary cohesive ends. The hybrid molecules are used to transform E. coli and the transformants contg. a DNA insert selected to provide a clone bank. - Clones contg. (II)-coding inserts are isolated (by testing with a (II)-specific probe), DNA extracted from them, inserted into plasmid vectors and recloned in E. coli. The clones having the DNA insert with the correct orientation and in the correct reading frame (as determined by restriction analysis and sequencing) are selected and ability to produce (I) confirmed by immunoassay. Also new is the complementary ds-DNA coding for (I) and E. coli clones contg. it.