Synthesis and expression of a gene from kringle-2 domain of tissue plasminogen activator in E. coli.

The DNA fragment corresponding to the tissue plasminogen activator (tPA) sequence 174-262 (Kringle-2 domain) has been synthesized by using the solid phase phosphotriester method. The Kringle-2 domain of human tPA was expressed in Escherichia coli by secretion into the periplasmic space using the Lpp-Lac promoter and PIN-III OmpA2 signal sequence. About two thirds of the expression product was secreted into the periplasmic space, and purified with ammonium sulfate fractionation, affinity chromatography on Lysine-Sepharose, and FPLC-Mono Q exchange chromatography. The amino acid composition observed from the Kringle-2 purified from E. coli is identical with that expected for the 174-262 fragment of human tPA. Radio binding assay shows that the recombinant Kringle-2 domain possesses the activity of fibrin binding.