We have isolated clones ofEscherichia coli strain K-12 that contain a hybrid pBR322 plasmid having a 4.2-kilobase insert of a DNA transcript of the mRNA of human plasminogen activator, urokinase. The bacterially produced enzyme has properties similar to those of urokinase from human fetal kidney cells. Both enzymes occur in discrete forms ranging from 32,000 to 150,000 daltons in size. They react with antibody to purified urokinase from human kidney cells, bind to a benzamidine-Sepharose column, and induce plasminogen-dependent lysis of a fibrin clot.For the dissolution of a blood clot, it is necessary to activate plasminogen to form the protease plasmin which then degrades the fibrin network of the clot. A plasminogen activator called "urokinase" was first observed in human urine in 1951 by Williams (1). Urokinase-like activity has been found in cultures of human kidney cells (2), and the material was subsequently shown to be immunologically indistinguishable from urokinase derived from urine. In urine, as well as in tissue culture, urokinases of two sizes are found; they are called "type S2" and "type Si,, and have molecular weights of 54,700 and 31,400, respectively (3, 4).Human urokinase can be used to treat acute thromboembolic events such as venous and arterial thrombosis, pulmonary embolism, intracardiac thrombosis, and systemic embolism. However, the high cost of isolation of urokinase from either tissue culture cells or urine limits the use of this enzyme as a therapeutic agent. If urokinase could be obtained from microorganisms by recombinant DNA technology, one might have a more economical method of production. This report describes the construction of hybrid bacterial plasmids that produce human urokinase in Escherichia coli.MATERIALS AND METHODS Bacterial Strain. E. coli K-12 strain x1776 (5) dapD8, minAl, minB2, supE42, A-, rfb-2, nalA25, oms-2, thyA57, metC65, oms-1, 29 (bioH-asd), cycB2, cycAl, hsdR) was provided by R. Curtiss III.DNA and Enzymes. Plasmid pBR322 was isolated as described by Ratzkin and Carbon (6). Reverse transcriptase was obtained from Life Sciences (St. Petersburg, FL). E. coli DNA polymerase I (the large fragment) and various restriction enzymes were purchased from New England BioLabs. Terminal transferase was obtained from P-L Biochemicals and nuclease S1 was purchased from Miles. Digestions with restriction enzymes were carried out under conditions recommended by the supplier.Antibody Against Urokinase. Anti-urokinase antibody synthesis was induced in New Zealand White rabbits by injection of purified urokinase (0.8 mg, type SI) emulsified in Freund's adjuvant. After three more injections of antigen over a period of2.5 months, the rabbits were bled. The antibody was purified by ammonium sulfate precipitation, followed by DEAE-cellulose chromatography and urokinase affinity chromatography (7). This antibody was checked for purity by electrophoresis (8) and for activity by the Ouchterlony test (9).Isolation ofmRNA from Human Fetal Kidney Cells. Human fetal kidney ce...
Strain SL100 is a gram-positive coccoid isolate prototype with an adhesin specific for gastric mucin and is representative of potentially pathogenic organisms obtained at biopsy from patients with gastric disorders. The urease of this isolate constitutes a significant fraction of the total cell protein, and the outcome of the purification strategy described herein suggests that it is associated with a cell wall fraction. The urease was purified 138-fold to apparent homogeneity, as indicated by gel electrophoresis, to a specific activity of 1,120 U/mg. The urease was unstable during purification in the absence of nickel, which is present in a metallocenter in other microbial ureases. When nickel sulfate was present during growth (5 M) and in buffers during sonication and purification (100 M), the urease was completely stable at room temperature during the purification procedure. The native urease was approximately 260 kDa and was composed of three subunits of 65 kDa and three subunits of 21 kDa. The purified urease was relatively stable in acid and retained most of its activity after incubation for 30 min at pH 1.3. The K m s for urease measured from whole cells and for the purified enzyme were 0.56 and 1.7 mM, respectively, indicating that some cell wall component(s) affects the affinity of the enzyme for urea. The V max s for urea hydrolysis measured from whole cells and for the purified enzyme were 8.1 and 1,120 mol/min/mg of protein, respectively. The kinetic parameters, relative abundance, and subunit composition are more similar to those of the ureases of Helicobacter than to those of the ureases of other microbial species. These similarities are consistent with an adaptation of this organism to colonization of the stomach and indicate that the urease may be a virulence factor during colonization.
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