title: Production and characterization of a novel tissue-type plasminogen activator derivative in Escherichia coli.
abstract: 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 Arg275 (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 Arg275 (the plasmin cleavage site) caused an increase in the catalytic enhancement by fibrin and a decrease of the interaction with plasminogen activator inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Pharmacological Research Laboratories, Fujisawa Pharmaceutical Co. Ltd., Osaka, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Saito
authors.forename: Y
authors.initials: Y
authors.lastname: Ishii
authors.forename: H
authors.initials: H
authors.lastname: Sasaki
authors.forename: M
authors.initials: M
authors.lastname: Hayashi
authors.forename: T
authors.initials: T
authors.lastname: Fujimura
authors.forename: Y
authors.initials: Y
authors.lastname: Imai
authors.forename: S
authors.initials: S
authors.lastname: Nakamura
authors.forename: S
authors.initials: S
authors.lastname: Suzuki
authors.forename: J
authors.initials: J
authors.lastname: Notani
authors.forename: T
authors.initials: T
authors.lastname: Asada
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004220:Disulfides / Q000378:metabolism; D004285:Dogs; D004926:Escherichia coli / Q000378:metabolism*; D005337:Fibrin / Q000378:metabolism / Q000494:pharmacology; D005343:Fibrinolytic Agents; D015870:Gene Expression; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011401:Promoter Regions, Genetic; D011489:Protein Denaturation; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000627:therapeutic use; D013927:Thrombosis / Q000188:drug therapy; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000627:therapeutic use; D014364:Tryptophan / Q000235:genetics; D001618:beta-Lactamases / Q000235:genetics
chemical_list: D004220:Disulfides; D005343:Fibrinolytic Agents; D011994:Recombinant Proteins; D014364:Tryptophan; D005337:Fibrin; C089949:D-K2P; D010959:Tissue Plasminogen Activator; D001618:beta-Lactamases