Secretion of active recombinant human tissue plasminogen activator derivatives in Escherichia coli

The DNA fragment coding for kringle 2 plus serine protease domains (K2S) of tissue plasminogen activator (tPA) was inserted into a phagemid vector, pC omb3HSS, In the recombinant vector, pComb3H-K2S, the K2S gene was fused to gpIII of phi M13 and linked to the OmpA signal sequence, The resulting gene , rK2S-gpIII, was inducibly expressed in Escherichia call XL-1 Blue, The pr otein was presented on the phage particle. To stop the expression of gpIII, a stop codon between K2S and the gpIII gene was inserted by site-directed mutagenesis, This mutated vector, MpComb3H-K2S, was transformed in XL-I Blu e. After induction with IPTG (isopropyl-beta -D-thiogalactopyranoside), rK2 S was found both in the periplasm as an inactive form of approximately 32% and in the culture supernatant as an active form of approximately 68%. The secreted form of rK2S was partially purified by ammonium sulfate (55%) prec ipitation, The periplasmic form was isolated from whole cells by chloroform extraction. The fibrin binding site of kringle 2 was demonstrated in all e xpressed versions (phage-bound, periplasmic, and secreted forms) using the monoclonal anti-kringle 2 antibody (16/B), Only the secreted form of rK2S r evealed a fibrinogen-dependent amidolytic activity with the specific activi ty of 236 IU/mug. No amidolytic activity of rK2S was observed in either the periplasmic or the phage-bound form. The secretion of rK2S as an active en zyme offers a novel approach for the production of the active-domain deleti on mutant tPA, rK2S, without any requirements for bacterial compartment pre paration and in vitro refolding processes. This finding is an important tec hnological advance in the development of large-scale, bacterium-based tPA p roduction systems.