Tissue plasminogen activator (tPA) is a polypeptide containing 527 amino acid residues (27,33) with a molecular mass of 72 kDa. The molecule is divided into five structural domains. Near the N-terminal region is a looped finger domain followed by a growth factor domain and the two domains kringle 1 and kringle 2. Both finger and kringle 2 domains bind specifically to the fibrin clots, thereby accelerating tPA protein activation of bound plasminogen. Next to the kringle 2 domain is the serine protease domain, which has the catalytic site located at the C terminus. This domain is responsible for converting plasminogen to plasmin, which is important for the homeostasis of fibrin formation and clot dissolution. The correct folding of tPA requires the correct pairing of 17 disulfide bridges in the molecule (1).

Clinically, tPA is a thrombolytic agent of choice for the treatment of acute myocardial infarction. It has the advantage of causing no side effects such as systemic hemorrhaging and fibrinogen depletion (7). Bowes melanoma cells were first used as a source of tPA production for therapeutic purposes (12). Since a consistent process efficiently producing high yields of highly purified protein is required for clinical use, the construction of full-length recombinant tPA (rtPA) progressed to mammalian cells. Chinese hamster ovary cells were transfected with the tPA gene to synthesize rtPA (8, 22). The recombinant product produced by a mammalian fermentation system was harvested from the culture medium. Attracted by simplicity and economy of production, investigators made a number of efforts in producing rtPA from bacteria, especially fromEscherichia coli (10, 13, 30). Numerous strategies have been proposed to overcome the problems of low yield and the formation of inclusion bodies, which result in misfolding and in an inactive enzyme. The other major criterion is to synthesize the smallest molecule which is still active, instead of full-length tPA.

Several deletion-mutant variants including kringle 2 plus serine protease (K2S) have been considered. However, the enzymatic activity of the recombinant K2S (rK2S) was obtained only when refolding of the purified inclusion bodies from the cytoplasmic compartment was achieved (16, 29). In order to avoid cumbersome refolding processes and periplasmic protein delivery, special bacterial expression systems were exploited (6, 31). Despite periplasmic expression of tPA, overexpression led to inactive aggregates, even in the relatively high-oxidizing conditions in the periplasm. The other possibility is synthesis of the recombinant protein as an extracellular component. Kipriyanov et al. (17) fused the PelB signal peptide to the N terminus of the single chain (ScFv) and were able to obtain an active recombinant product from E. coli culture supernatant. Recently, the expression of certain recombinant antibody fragments in L forms ofProteus mirabilis was described (28). As the bacterial cells lack periplasmic space, the active recombinant antibody fragment could be directly harvested from the culture supernatant. However, the properties of an extracellular form of the heterologous protein may be distinct from those of the periplasmic accumulated form.

In this study, we describe the production of extracellular and periplasmic forms of K2S in E. coli. In a recent report, the enzymatic properties of phage-displayed kringle 5 with the plasminogen serine protease have been successfully demonstrated (20). Since the cysteine number of this protein is similar to that of K2S, we have evaluated whether the phage-displayed rK2S exerts protease activity. Moreover, we applied a novel strategy to secrete rK2S into the culture supernatant of E. coli. The phage-displayed rK2S and the periplasmic and secreted forms were compared with respect to fibrinogen-dependent serine protease activity.