Purification methods and uses thereof
OwnerDavid Saul, Roy Danielinventors
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Abstract
This invention relates to the use of thermophilic proteases in combination with mesophilic enzymes, including such use in improved methods for isolation, purification, diagnostic, analytical and manufacturing techniques. In particular, the uses and methods of the invention will facilitate improved purification, diagnostic, analytical and manufacturing techniques, especially those in the field of molecular biology, by providing means of removing contamination and/or undesired constituents, and/or by simplifying the techniques.
Claims58
A use of at least one thermophilic protease and one or more mesophilic enzyme in combination in the removal of at least one contaminant from a composition, wherein said contaminant is a substrate of one or more mesophilic enzyme, and wherein said composition is not a nucleic acid sample comprising nucleic acid for testing.
A use of at least one thermophilic protease and one or more mesophilic enzyme that does not exhibit activity effective to remove cell walls in combination in the removal of at least one contaminant from a composition, wherein said contaminant is a substrate of said one or more mesophilic enzyme.
A method for the removal of at least one contaminant from a composition, wherein said contaminant is a substrate of one or more mesophilic enzyme, said method comprising the steps of:
- adding to the composition said one or more mesophilic enzyme and at least one thermophilic protease;
- and
- maintaining said composition at a first temperature or temperature range at which said one or more mesophilic enzyme has enzymatic activity, but at which said at least one thermophilic protease has minimal or no proteolytic activity;
- and
- maintaining said composition at a second temperature or temperature range at which said at least one thermophilic protease has proteolytic activity;
- and
- maintaining said composition at a temperature suitable for use;
- wherein said one or more mesothermic enzyme does not exhibit activity effective to remove cell walls.
A method for the removal of at least one contaminant from a composition, wherein said contaminant is a substrate of one or more mesophilic enzyme, said method comprising the steps of:
- adding to the composition said one or more mesophilic enzyme and at least one thermophilic protease;
- and
- maintaining said composition at a first temperature or temperature range of from 40° C. to about 65° C. at which said one or more mesophilic enzyme has enzymatic activity;
- and
- maintaining said composition at a second temperature or temperature range at which said at least one thermophilic protease has proteolytic activity;
- and
- maintaining said composition at a temperature suitable for use.
A method for the removal of nucleic acid from a composition, said method comprising the steps of:
- adding to the composition one or more mesophilic nucleic acid-degrading enzyme and at least one thermophilic protease;
- and
- maintaining said composition at a first temperature or temperature range at which said one or more mesophilic nucleic acid-degrading enzyme has enzymatic activity, but at which said at least one thermophilic protease has minimal or no proteolytic activity;
- and
- maintaining said composition at a second temperature or temperature range at which said at least one thermophilic protease has proteolytic activity;
- and
- maintaining said composition at a temperature suitable for use.
A method for purifying one or more working solution for nucleic acid diagnostic techniques in a closed-tube system, said method comprising the steps of:
- adding to said one or more working solution one or more nuclease and at least one thermophilic protease capable of degrading potentially contaminating nucleic acid and/or nucleic acid degrading enzymes in said working solution;
- and
- storing said one or more working solution at a preferred temperature until required for use;
- and
- completing purification prior to using said one or more working solution by raising the temperature of said one or more working solution;
- and
- cooling said one or more working solution for use, the method characterised by the use of both a nuclease and a thermophilic protease having differing temperature activity profiles.
Dependencies are inferred from the claim text.