An anti-human immunodeficiency virus (anti-HIV) protein capable of inhibiting HIV-1 infection and replication has been isolated and purified to homogeneity from Trichosanthes krilowU. This protein, TAP 29 (Trichosanthes anti-HIV protein, 29 kDa), is distinct from trichosanthin [also known as GLQ 223 (26 kDa)] in size, N-terminal amino acid sequence, and cytotoxicity. In addition to three conservative

A recombinant plasminogen activator with high fibrin affinity and specificity was expressed by transfecting hybridoma cells with a plasmid that combines sequence coding for low molecular mass (32 kDa) single-chain urokinasetype plasminogen activator [scuPA(32kDa)J and anti-fibrin monoclonal antibody 59D8. The expression of the recombinant molecule [r-scuPA(32kDa)-59D8] was optimized by replacing the 3' untranslated region (initially that of high molecular mass scuPA) in the plasmid with the 3' untranslated region of either 13-globin or mouse immunoglobulin. This modification resulted in a >100-fold improvement in the level of protein expression.The 103-kDa r-scuPA(32kDa)-59D8 protein displayed catalytic activity indistinguishable from that of high molecular mass scuPA and fibrin binding comparable to that of native antibody 59D8. r-scuPA(32kDa)-59D8 was 6 times more potent than high molecular mass scuPA in lysing a human plasma clot in vitro and was 20 times more potent than high molecular mass scuPA in the rabbit jugular vein model of thrombolysis.Molecules of this type may serve as prototypes for highly specific, antibody-targeted enzymes suitable for human use.Acute thrombotic occlusion of a major epicardial coronary artery results in myocardial infarction, the single most common cause of death in industrialized societies. The use of thrombolytic therapy in patients with acute myocardial infarction has resulted in a significant reduction in mortality (1-3). At its present stage of development, however, thrombolytic therapy is limited by (i) significant bleeding at high doses (intracranial hemorrhage causes stroke or death in 0.1-0.5% of patients who receive plasminogen activators)(1-4), (ii) the failure to restore blood flow in 20%o of patients or the fact that thrombotic reocclusion after cessation of therapy occurs in 15-25% of patients (5), and (iii) the lag between initiation of therapy and clot lysis, which averages about 60 min (5).These limitations have prompted generation (by recombinant DNA technology) of hundreds of plasminogen activator mutants, which have produced, at best, only modest improvements in thrombolytic efficacy. Most investigators have either rearranged (or duplicated or deleted) various plasminogen activator functional domains or altered their posttranslational modification (6-10). We and others (11-16) have pursued an alternative strategy, the generation ofchemical conjugates or recombinant molecules with domains for both plasminogen activator activity and high-affinity fibrin binding [conferred by a monoclonal antibody (17) that binds to fibrin, the principal component of a thrombus, but not fibrinogen, its circulating precursor]. Here we report the generation and characterization (in vitro and in vivo) of a recombinant molecule that is fibrin selective by two different mechanisms. It combines a high-affinity anti-fibrin antibody, 59D8, with a low molecular mass (32 kDa) single-chain urokinase-type plasminogen activator [scuPA(32kDa)], a fibrin-selective plasminogen activato...

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This study involved the construction of hybrid plasmids to produce heat-stable enterotoxin type II of Escherichia coli (STb). The translation of the open reading frame for the STb gene estA was demonstrated in several ways. Studies using in vivo labeling with [35S]cysteine demonstrated a radiolabeled protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the expected molecular weight of 5,000 for toxin STb. Insertion of translational or transcriptional termination signals into the Bglll site of the estA gene blocked the expression of estA. The estA gene was cloned into high-expression vector pKC30 downstream from the strong Pl promoter. Northern (RNA) blotting assays revealed a 10to 20-fold increase in mRNA produced by strain C600F(pKC30STb) over other STb-producing strains, compared with little or no increase in enterotoxin activity demonstrated by bioassay. The estA gene, with its own promoter and Shine-Delgarno region and a portion of the sequence for the signal peptide deleted, was also inserted under the control of the tac promoter. Even after induction of the tac promoter by addition of isopropyl-,I-D-thiogalactopyranoside, no biologic enterotoxin activity could be identified. Neutralizing antibodies to STb were produced in rabbits by using either a purified OmpF-STb-pi-galactosidase fusion protein or a 19-amino-acid synthetic STb peptide coupled to * Corresponding author. provided by C. H. Lee (15). All of the plasmids used are described in Table 1. The host strains were E. coli MH3000 and TK1046 for pORF1 derivatives (41). C600F, N99CI+, and N5151 were used as hosts for pKC30 and its derivatives (2, 31). E. coli JM103 was used as the host for pDR540 and its subclones (42). E. coli C strain 3456 was used as the host for pCHL6. P3, a wild-type strain of E. coli containing the gene for enterotoxin STb, estA, and 10405, a wild-type E. coli strain without estA, were used as controls in the bioassays. All of the strains used are listed in Table 2. Media. Bacteria were grown in tryptone yeast extract medium or minimal medium A supplemented with 0.25% glucose or 0.20% lactose. Clones were screened for lactose fermentation on MacConkey lactose plates or on 5-bromo-4-chloro-3-indolyl-4-D-galactopyranoside plates with or without isopropyl- ,-D-thiogalactopyranoside (IPTG). In vivo radiolabeling was performed in modified minimal medium A (0.004 M dipotassium phosphate, 0.0022 M potassium biphosphate, 0.0017 M trisodium citrate, 0.0076 M ammonium sulfate, 0.008 M magnesium sulfate, 0.01% yeast extract, 5% glucose, required growth factors). Ampicillin was used at 50 ,ug/ml, and tetracycline was used at 20 ,ug/ml. Materials. Enzymes were purchased from New England BioLabs, Inc., or Boehringer Mannheim Biochemicals and used as recommended by the suppliers. 5-Bromo-4-chloro-3-indolyl-4-D-galactopyranoside, IPTG, and p-aminophenyl-,B-D-thiogalactosidase were purchased from Sigma Chemical Co. o-Nitrophenyl-,B-D-galactopyranoside was supplied by Boehringer Mannheim Biochemicals. Oligonucleotides were sy...