Biological entities and the use thereof

Abstract

The present invention provides engineered enzymes generated from protein scaffolds combined with Specificity Determining Regions, the production thereof and the use of said engineered enzymes for research, nutritional care, personal care and industrial purposes.

1 . A recombinant engineered enzyme with catalytic activity of defined specificity, characterized by a combination of the following components:
 (a) a protein scaffold capable of catalyzing at least one protein cleavage reaction on at least one target substrate and being a serine protease of the structural class S8, and   (b) one or more specificity determining regions (SDRs), wherein the SDRs are peptide sequences inserted into the protein scaffold at one or more positions that correspond structurally or by amino acid sequence homology to the regions 6-17, 25-29, 47-55, 59-69, 101-111, 117-125, 129-137, 139-154, 158-169, 185-195 and 204-225 in subtilisin E from  Bacillus subtilis  having the amino acid shown in SEQ ID NO:7, wherein the inserted SDRs enable the resulting engineered protein to discriminate between at least one target substrate and one or more different substrates.   
     
     
         2 . The recombinant engineered enzyme of  claim 1 , wherein the SDRs (b) have a length of less than 50 amino acid residues. 
     
     
         3 . The recombinant engineered enzyme of  claim 1 , wherein the SDRs (b) have a length between two and 20 amino acid residues.
 The above recombinant engineered enzyme, wherein the SDRs (b) have a length between two and ten amino acid residues.   
     
     
         5 . The recombinant engineered enzyme of claim  4 , wherein the SDRs (b) have a length between three and eight amino acid residues. 
     
     
         6 . The recombinant engineered enzyme of  claim 2 , wherein the number of SDRs is at least one. 
     
     
         7 . The engineered enzyme of  claim 6 , wherein the number of SDRs is more than one. 
     
     
         8 . The recombinant engineered enzyme of  claim 6 , wherein the number of SDRs is between two and eleven. 
     
     
         9 . The recombinant engineered enzyme of  claim 6 , wherein the number of SDRs is between two and six. 
     
     
         10 . The recombinant engineered enzyme of  claim 1 , wherein the protein scaffold (a) is encoded by a gene of viral origin. 
     
     
         11 . The recombinant engineered enzyme of  claim 1 , wherein the protein scaffold (a) is encoded by a gene of prokaryotic origin. 
     
     
         12 . The recombinant engineered enzyme of  claim 1 , wherein the protein scaffold (a) is encoded by a gene of eukaryotic origin. 
     
     
         13 . The recombinant engineered enzyme of  claim 1 , wherein the protein scaffold (a) is comprised of one or more polypeptides derived from the same or different native enzymes. 
     
     
         14 . The recombinant engineered enzyme of  claim 1 , wherein the protein scaffold (a) is comprised of one or more polypeptides derived from the same or different native mammalian enzymes. 
     
     
         15 . The recombinant engineered enzyme of  claim 14 , wherein the mammalian enzymes are human enzymes. 
     
     
         16 . The engineered enzyme of  claim 1 , wherein the SDRs are located at one or more positions selected from the group of positions that correspond structurally or by amino acid sequence homology to the regions 59-69, 101-111, 129-137, 158-169 and 204-225 in subtilisin E from  Bacillus subtilis  having the amino acid shown in SEQ ID NO:7. 
     
     
         17 . A fusion protein which is comprised of at least one engineered enzyme of  claim 1  and at least one further proteinaceous component. 
     
     
         18 . The fusion protein of  claim 17 , wherein the further proteinaceous component is selected from the group consisting of binding domains, receptors, antibodies, regulation domains, pro-sequences, and fragments thereof. 
     
     
         19 . A fusion protein which is comprised of at least one engineered enzyme of  claim 1  and at least one further functional component. 
     
     
         20 . The fusion protein of  claim 19 , wherein the functional component is selected from the group consisting of polyethyleneglycols, carbohydrates, lipids, fatty acids, nucleic acids, metals, metal chelates, and fragments or derivatives thereof. 
     
     
         21 . A composition comprising one or more engineered enzymes of  claim 1 . 
     
     
         22 . A composition comprising a fusion protein of  claim 17 . 
     
     
         23 . A composition comprising a fusion protein of  claim 19 . 
     
     
         24 . The composition of  claim 21 , which is a composition selected from the group consisting of research composition, nutritional composition, cleaning composition, food additive composition, disinfection composition, cosmetic composition or composition for personal care. 
     
     
         25 . The composition of  claim 22 , which is a composition selected from the group consisting of research composition, nutritional composition, cleaning composition, food additive composition, disinfection composition, cosmetic composition or composition for personal care. 
     
     
         26 . The composition of  claim 23 , which is a composition selected from the group consisting of research composition, nutritional composition, cleaning composition, food additive composition, disinfection composition, cosmetic composition or composition for personal care. 
     
     
         27 . The composition of  claim 21 , which further comprises optional components selected from the group consisting of acceptable carrier(s) and auxiliary agent(s). 
     
     
         28 . The composition of  claim 22 , which further comprises optional components selected from the group consisting of acceptable carrier(s) and auxiliary agent(s). 
     
     
         29 . The composition of  claim 23 , which further comprises optional components selected from the group consisting of acceptable carrier(s) and auxiliary agent(s). 
     
     
         30 . A recombinant engineered enzyme with catalytic activity of defined specificity, characterized by a combination of the following components:
 (a) a protein scaffold capable of catalyzing at least one protein cleavage reaction on at least one target substrate and being an aspartic protease of the structural class A1, and   (b) one or more specificity determining regions (SDRs), wherein the SDRs are peptide sequences inserted into the protein scaffold at one or more positions that correspond structurally or by amino acid sequence homology to the regions 6-18, 49-55, 74-83, 91-97, 112-120, 126-137, 159-164, 184-194, 242-247, 262-267 and 277-300 in human pepsin having the amino acid sequence shown in SEQ ID NO:11, wherein the inserted SDRs enable the resulting engineered protein to discriminate between at least one target substrate and one or more different substrates.   
     
     
         31 . The engineered enzyme of  claim 30 , wherein the SDRs are located at one or more positions selected from the group of positions that correspond structurally or by amino acid sequence homology to the regions 10-15, 75-80, 114-118, 130-134, 186-191 and 280-296 in human pepsin having the amino acid sequence shown in SEQ ID NO:11. 
     
     
         32 . A recombinant engineered enzyme with catalytic activity of defined specificity, characterized by a combination of the following components:
 (a) a protein scaffold capable of catalyzing at least one protein cleavage reaction on at least one target substrate and being a cysteine protease of the structural class C14, and   (b) one or more specificity determining regions (SDRs), wherein the SDRs are peptide sequences inserted into the protein scaffold at one or more positions that correspond structurally or by amino acid sequence homology to the regions 78-91, 144-160, 186-198, 226-243 and 271-291 in human caspase 7 having the amino acid sequence of SEQ ID NO:14, wherein the inserted SDRs enable the resulting engineered protein to discriminate between at least one target substrate and one or more different substrates.   
     
     
         33 . The engineered enzyme of  claim 32 , wherein the SDRs are located at one or more positions selected from the group of positions that correspond structurally or by amino acid sequence homology to the regions 80-86, 149-157, 190-194 and 233-238 of human caspase 7 having the amino acid sequence of SEQ ID NO:14. 
     
     
         34 . A recombinant engineered enzyme with catalytic activity of defined specificity, characterized by a combination of the following components:
 (a) a protein scaffold capable of catalyzing at least one protein cleavage reaction on at least one target substrate and being derived from a protease selected from the group consisting of a serine protease of the structural class S1, S8, S11, S21, S26, S33 and S51, a cysteine protease of the structure class C1, C2, C4, C10, C14, C19, C47, C48 and C56, an aspartic protease of the structural class A1, A2 and A26, and a metalloprotease of the structural class M4 and M10, and   (b) one or more specificity determining regions (SDRs) located at sited in the protein scaffold that enable the resulting engineered protein to discriminate between at least one target substrate and one or more different substrates and wherein the SDRs are essentially synthetic peptide sequences.