Methods for the analysis of non-proteinaceous components using a protease from a Bacillus strain

Abstract

This invention relates to a method for the analysis of a (at least one) target non-proteinaceous component of a mixture of non-proteinaceous and proteinaceous components derived from a biological sample using a protease from a Bacillus strain. The invention further relates to a method for the analysis of a (at least one) target nucleic acid component of a mixture of non-proteinaceous components, which comprise nucleic acids, and proteinaceous components whereby the mixture is derived from a biological sample comprising the steps of incubating the mixture with a (at least one) protease from a Bacillus strain, optionally amplifying the (at least one) target nucleic acid component, and determining or detecting the (at least one) target nucleic acid component.

1 . A method for the analysis of a target non-proteinaceous component of a mixture of non-proteinaceous and proteinaceous components derived from a biological sample comprising the step of incubating the mixture with a protease having an amino acid sequence which is at least 80% identical to the amino acid sequence of the protease subtilisin 147 from  Bacillus lentus.    
     
     
         2 . The method according to  claim 1   
       wherein 
 the amino acid sequence of the protease is identical to the amino acid sequence of the protease subtilisin 147 from  Bacillus lentus.    
 
     
     
         3 . The method according to  claim 1   
       wherein 
 the amino acid sequence of protease is the amino acid sequence SEQ ID NO 1, a proteolytical derivative thereof having protease activity or the amino acid sequence SEQ ID NO 2.  
 
     
     
         4 . The method according to  claim 1  wherein 
 the amino acid sequence of the protease is encoded by the nucleic acid sequence SEQ ID NO 3, a part thereof or a degenerate version of the nucleic acid sequence SEQ ID NO 3.  
 
     
     
         5 . The method according to  claim 1   wherein the biological sample is a fluid from the human or animal body.    
     
     
         6 . The method according to  claim 1  wherein 
 the biological sample is blood, blood plasma, blood serum or urine.  
 
     
     
         7 . The method according to  claim 1   
       wherein 
 the biological sample comprises bacterial cells, eukaryotic cells, viruses or mixtures thereof.  
 
     
     
         8 . The method according to  claim 1 ,  
       wherein 
 after the incubation step the target non-proteinaceous component is bound to a material with an affinity thereto, optionally washed and optionally released from the material with an affinity thereto.  
 
     
     
         9 . The method according to  claim 1   
       wherein 
 the non-proteinaceous component comprises a nucleic acid.  
 
     
     
         10 . The method according to  claim 9   
       wherein 
 the nucleic acid comprises DNA or RNA or both.  
 
     
     
         11 . A method for the analysis of a target nucleic acid component of a mixture comprising the target nucleic acid component, and a proteinaceous component whereby the mixture is derived from a biological sample, which method comprising the steps of 
 a) incubating the mixture with a protease having an amino acid sequence which is at least 80% identical to the amino acid sequence of the protease subtilisin 147 from  Bacillus lentus,      b) optionally amplifying the target nucleic acid component, and    c) determining or detecting the target nucleic acid component.    
     
     
         12 . The method according to  claim 11   
       wherein 
 the amino acid sequence of the protease is identical to the amino acid sequence of the protease subtilisin 147 from  Bacillus lentus.    
 
     
     
         13 . The method according to  claim 11   
       wherein 
 the amino acid sequence of protease is the amino acid sequence SEQ ID NO 1, a proteolytical derivative thereof having protease activity or the amino acid sequence SEQ ID NO 2.  
 
     
     
         14 . The method according to  claim 11   wherein the amino acid sequence of the protease is encoded by the nucleic acid sequence SEQ ID NO 3, a part thereof or a degenerated version of the nucleic acid sequence SEQ ID NO 3.    
     
     
         15 . The method according to  claim 11   
       wherein 
 the biological sample is a fluid from the human or animal body.  
 
     
     
         16 . The method according to  claim 11   
       wherein 
 the biological sample is blood, blood plasma, blood serum or urine.  
 
     
     
         17 . The method according to  claim 11   
       wherein 
 the target nucleic acid component comprises DNA or RNA or both.  
 
     
     
         18 . The method according to  claim 17   wherein the DNA or RNA or both is derived from a virus or a microorganism.    
     
     
         19 . The method according to  claim 18   
       wherein 
 the virus is hepatitis B virus, hepatitis C virus or the human immunodeficiency virus.  
 
     
     
         20 . The method according to  claim 11   
       wherein 
 the target nucleic acid component is amplified with the polymerase chain reaction.  
 
     
     
         21 . The method according to  claim 11   
       wherein 
 after step a) the target nucleic acid component is bound to a material with an affinity to nucleic acids, optionally washed and optionally released from the material.  
 
     
     
         22 . The method according to  claim 21   
       wherein 
 the material with an affinity to nucleic acids comprises a material with a silica surface.  
 
     
     
         23 . The method according to  claim 22   wherein the material with a silica surface is a glass.    
     
     
         24 . The method according to  claim 21   wherein the material with an affinity to nucleic acids is a composition comprising magnetic glass particles.    
     
     
         25 . The method according to any of the claims  1  to 24, wherein the analysis is a diagnosis of a disease or a pathogen.  
     
     
         26 . A kit comprising a protease having an amino acid sequence, which is at least 80% identical to the amino acid sequence of the protease subtilisin 147 from  Bacillus lentus.    
     
     
         27 . The kit according to  claim 26   
       wherein 
 the amino acid sequence of the protease is identical to the amino acid sequence of the protease subtilisin 147 from  Bacillus lentus.    
 
     
     
         28 . The kit according to  claim 26   
       wherein 
 the amino acid sequence of protease is the amino acid sequence SEQ ID NO 1, a proteolytical derivative thereof having protease activity or the amino acid sequence SEQ ID NO 2.  
 
     
     
         29 . The kit according to  claim 26   
       wherein 
 the amino acid sequence of the protease subtilisin 147 is encoded by the nucleic acid sequence SEQ ID NO 3, a part thereof or a degenerated version of the nucleic acid sequence SEQ ID NO 3.  
 
     
     
         30 . The kit according to  claim 26   wherein it additionally contains a material with an affinity to nucleic acids.    
     
     
         31 . The kit according to  claim 30   
       wherein 
 the material with an affinity to nucleic acids comprises a material with a silica surface.  
 
     
     
         32 . The kit according to  claim 31   
       wherein 
 the material with a silica surface is a glass.  
 
     
     
         33 . The kit according to  claim 26   
       wherein 
 the material with an affinity to nucleic acids is a composition comprising magnetic glass particles.  
 
     
     
         34 . The kit according to  claim 32   
       wherein 
 the kit additionally comprises a lysis buffer, a washing buffer and an elution buffer.  
 
     
     
         35 . A composition comprising a protease which is at least 80% identical to the amino acid sequence of the protease subtilisin 147 from  Bacillus lentus , in a solution 10 mM Tris acetate pH 5.5, 5 mM calcium chloride, 5 mM calcium acetate, 1 mM EDTA, and 50% (V/V) Glycerin.  
     
     
         36 . The composition according to  claim 35   
       wherein 
 the amino acid sequence of the protease is identical to the amino acid sequence of the protease subtilisin 147 from  Bacillus lentus.    
 
     
     
         37 . The composition according to  claim 35   
       wherein 
 the amino acid sequence of protease is the amino acid sequence SEQ ID NO 1, a proteolytical derivative thereof having protease activity or the amino acid sequence SEQ ID NO 2.  
 
     
     
         38 . The composition according to  claim 35   
       wherein 
 the amino acid sequence of the protease subtilisin 147 is encoded by the nucleic acid sequence SEQ ID NO 3, a part thereof or a degenerated version of the nucleic acid sequence SEQ ID NO 3.