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Production and Characterization of a Novel Tissue‐Type Plasminogen Activator Derivative in Escherichia coli
Author(s) -
Saito Yoshimasa,
Ishii Yoshinori,
Sasaki Hitoshi,
Hayashi Masako,
Fujimura Takao,
Imai Yuko,
Nakamura Sachiko,
Suzuki Shingo,
Notani Joji,
Asada Takashi,
Horiai Haruo,
Kobayashi Masakazu,
Niwa Mineo
Publication year - 1994
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp00029a004
Subject(s) - escherichia coli , plasminogen activator , plasmin , microbiology and biotechnology , tissue plasminogen activator , mutant , chemistry , biochemistry , expression vector , protease , recombinant dna , biology , enzyme , gene , endocrinology
We have created a novel thrombolytic agent by the combination of mutation with partial deletion of tissue‐type plasminogen activator (t‐PA). We constructed Escherichia coli expression vectors for (i) native t‐PA (nt‐PA) and its derivatives; (ii) K1K2P, consisting of kringle 1 (K1), kringle 2 (K2), and protease (P) domains; (iii) K2P, consisting of K2 and P domains; (iv) D‐nt‐PA, (v) D‐K1K2P; and (vi) D‐K2P. The latter three are point mutants of nt‐PA, K1K2P, and K2P, respectively, in which Arg 275 (number corresponds to that of nt‐PA) has been mutated to Asp. The production of nt‐PA and its derivatives was remarkably improved by (i) removal of the 3× noncoding region of nt‐PA cDNA from expression vectors and (ii) expression in mutant E. coli derived from E. coli HB101, which is insensitive to heat‐shock inductions. The proteins produced were precipitated as insoluble aggregates in the cells and were renatured to active forms by extraction with 8 M urea followed by dialysis against a redox buffer containing GSH and GSSG. The renaturation yield depended on the pH of the buffer and the number of disulfide bonds of the proteins (nt‐PA ≪ K1K2P < K2P). The mutation of Arg 275 (the plasmin cleavage site) caused an increase in the catalytic enhancement by fibrin and a decrease of the interaction with plasminogen activator inhibitors. Among them, D‐K2P was selected as a candidate for a new thrombolytic agent because of (i) its facility of renaturation and purification to homogeneity and (ii) its high catalytic enhancement by fibrin, as compared with nt‐PA or other derivatives produced by the same system. It was produced in large amounts and characterized by structural analysis, as well as by biological and pharmacological activities. The disulfide linkages of D‐K2P were found to be identical to those of nt‐PA (Bowes) on the kringle 2 and protease domains. Although its specific activity is approximately one‐fourth that of nt‐PA (Bowes), it shows higher fibrin acceleration and a longer half‐life in blood than nt‐PA (Bowes). In the preliminary pharmacological studies, D‐K2P exhibited more potential thrombolytic effect than recombinant nt‐PA (alteplase) by both bolus and infusional administrations in the rabbit thrombus model. It was also more effective in canine coronary thrombosis. These results may indicate that plasma half‐life and fibrin acceleration play important roles in the thrombolytic agent.