High yield production and purification of recombinant staphylokinase for thrombolytic therapy.
Recombinant plasmids were constructed in which the signal sequence of the sak42D and the sakSTAR staphylokinase genes were replaced by an ATG start codon and which express staphylokinase under the control of a tac promoter and two Shine-Dalgarno sequences in tandem. Induction of transfected E. coli TGl cells in a bacterial fermentor produced intracellular staphylokinase representing 10 to 15% of total cell protein. Gram quantities of highly purified recombinant staphylokinase were obtained from cytosol fractions by chromatography, at room temperature, on SP-Sepharose and on phenyl-Sepharose columns, with yields of 50 to 70 percent. The material, at a dose of 4 mg/kg, did not produce acute reactions or affect body weight in mice. Intravenous administration of 10 mg SakSTAR over 30 minutes in five patients with acute myocardial infarction induced complete coronary artery recanalization, without associated fibrinogen degradation. However, neutralizing antibodies appeared in the plasma of all patients within 12 to 20 days. Thus, the present expression and purification method for recombinant staphylokinase yields large amounts of highly purified mature protein (approximately 200 mg per liter fermentation broth) suitable for a more detailed clinical investigation of its potential as a thrombolytic agent.
Schlott
B
B
Institute for Molecular Biotechnology, Jena, Germany.
Hartmann
M
M
Gührs
K H
KH
Birch-Hirschfeid
E
E
Pohl
H D
HD
Vanderschueren
S
S
Van de Werf
F
F
Michoel
A
A
Collen
D
D
Behnke
D
D
D001483:Base Sequence; D002852:Chromatography, Ion Exchange / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods; D003062:Codon; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004795:Enzyme Stability; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D008666:Metalloendopeptidases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D010957:Plasmids; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D015912:Thrombolytic Therapy
D003062:Codon; D009838:Oligodeoxyribonucleotides; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus