Methods of making pro-urokinase mutants
AssigneeThrombolytic Science, Inc.
InventorsPaolo Sarmientos, Massimiliano Pagani
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Abstract
Methods are provided for producing non-glycosylated, single-chain and two-chain pro-urokinase (pro-UK) mutants. The methods include cultivating a specific E. coli type B strain that has been transformed with specific plasmids carrying a cDNA sequence that encodes pro-UK mutants and carries specific promoter sequences. Products produced by the methods have medical use for thrombolysis performed while sparing hemostatic clots, e.g., for particular applications such as after a stroke or heart attack.
Claims24
A method of preparing a pro-urokinase (“pro-UK”) mutant polypeptide, the method comprising
- (a) obtaining a nucleic acid molecule that encodes a pro-UK mutant polypeptide;
- (b) inserting the nucleic acid molecule into a pET29a expression plasmid comprising a phage T7 promoter and Shine-Dalgamo sequence;
- (c) transforming E. coli type B strain bacteria BL21/DE3 RIL with the expression plasmid;
- (d) culturing the transformed bacteria for a time and under conditions sufficient to enable the bacteria to express pro-UK mutant polypeptide;
- and
- (e) isolating the pro-UK mutant polypeptide from the transformed bacteria.
A purified culture of E. coli type B strain bacteria BL21DE3 RIL, wherein bacteria in the culture comprise an expression plasmid encoding a pro-urokinase flexible loop mutant polypeptide.
A method of preparing a pro-urokinase (“pro-UK”) mutant polypeptide, the method comprising
- (a) obtaining a transformed bacteria, wherein the bacteria is an E. coli type B strain bacteria BL21DE3 RIL transformed with a pET29a expression plasmid comprising a phage T7 promoter, a Shine-Dalgamo sequence, and a nucleic acid molecule that encodes a pro-UK mutant polypeptide;
- (b) culturing the transformed bacteria for a time and under conditions sufficient to enable the bacteria to express pro-UK mutant polypeptide;
- and
- (c) isolating the pro-UK mutant polypeptide from the transformed bacteria.
Dependencies are inferred from the claim text.