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 S1, and   (b) one or more specificity determining regions (SDRs), wherein the SDRs are peptide sequences that are inserted into the protein scaffold at one or more positions that correspond structurally or by amino acid sequence homology to the regions 18-25, 54-63, 73-86, 148-156, 165-171 and 194-204 in human trypsin I having the amino acid sequence shown in SEQ ID NO: 1, 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 2 , wherein the SDRs (b) have a length between two and 20 amino acid residues. 
     
     
         4 . The recombinant engineered enzyme of  claim 3 , 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 recombinant 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) 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 being 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 being 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 - 28 . (canceled) 
     
     
         29 . The recombinant engineered enzyme of  claim 1 , further comprising SDRs located at one or more positions selected from the group of positions that correspond structurally or by amino acid sequence homology to the regions 38-48, and 122-130 in human trypsin I having the amino acid sequence shown in SEQ ID NO: 1. 
     
     
         30 . The recombinant 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 20-23, 57-60, 76-83, 150-153, 167-169 and 197-201 in human trypsin I having the amino acid sequence shown in SEQ ID NO: 1. 
     
     
         31 . The recombinant engineered enzyme of  claim 1 , wherein the protein scaffold (a) is derived from the serine protease trypsin. 
     
     
         32 . The recombinant engineered enzyme of  claim 31 , wherein the serine protease trypsin is human trypsin I having the amino acid sequence shown in SEQ ID NO:1 or a derivative thereof. 
     
     
         33 . The recombinant engineered enzyme of  claim 31 , wherein the serine protease trypsin has amino acid sequence SEQ ID NO: 1 and comprises one or more of the amino acid substitutions selected from the group consisting of E56G, R78W, Y131F, A146T and C183R. 
     
     
         34 . The recombinant engineered enzyme of claim  28 , which has at least one of two SDRs located in the scaffold, a first SDR having a length of up to 6 amino acids and being inserted between residues 42 and 43, and a second SDR having a length of up to 5 amino acids and being inserted between residues 123 and 124, the numbering being relative to human trypsin I having the amino acid sequence shown in SEQ ID NO: 1. 
     
     
         35 . The recombinant engineered enzyme of  claim 34 , which comprises one of the peptide sequences of the following group: SEQ ID NO: 72, 78, 79, 80, 84, 85, 86, 87, 88, and 89 inserted as the first SDR between residues 42 and 43. 
     
     
         36 . The recombinant engineered enzyme of  claim 34 , which comprises one of the peptide sequences of the following group: SEQ ID NO: 73, 81, 82, 83, 90, 91, 92, 93, 94, and 95 inserted as the second SDR between residues 123 and 124. 
     
     
         37 . The recombinant engineered enzyme of  claim 31 , which comprises an amino acid sequence selected from the group consisting of SEQ ID NO:74 and SEQ ID NO:75. 
     
     
         38 - 44 . (canceled) 
     
     
         45 . A fusion protein which is comprised of at least one engineered enzyme of  claim 1  and at least one further proteinacious component. 
     
     
         46 . The fusion protein of  claim 45 , wherein the further proteinacious component is selected from the group consisting of binding domains, receptors, antibodies, regulation domains, pro-sequences, and fragments thereof. 
     
     
         47 . A fusion protein which is comprised of at least one engineered enzyme of  claim 1  and at least one further functional component. 
     
     
         48 . The fusion protein of  claim 47 , wherein the functional component is selected from the group consisting of polyethylenglycols, carbohydrates, lipids, fatty acids, nucleic acids, metals, metal chelates, and fragments or derivatives thereof. 
     
     
         49 - 71 . (canceled) 
     
     
         72 . A composition comprising one or more engineered enzymes of  claim 1 . 
     
     
         73 . A composition comprising a fusion protein of  claim 47 . 
     
     
         74 . A composition comprising a fusion protein of  claim 45 . 
     
     
         75 . The composition of  claim 72 , which is a composition selected from the group consisting of research composition, nutritional composition, cleaning composition, food additive composition, desinfection composition, cosmetic composition and composition for personal care. 
     
     
         76 . The composition of  claim 73 , which is a composition selected from the group consisting of research composition, nutritional composition, cleaning composition, food additive composition, desinfection composition, cosmetic composition and composition for personal care. 
     
     
         77 . The composition of  claim 74 , which is a composition selected from the group consisting of research composition, nutritional composition, cleaning composition, food additive composition, desinfection composition, cosmetic composition and composition for personal care. 
     
     
         78 . The composition of  claim 72 , which further comprises optional components selected from the group consisting of a pharmaceutically acceptable carrier(s) and auxiliary agent(s). 
     
     
         79 . The composition of  claim 73 , which further comprises optional components selected from the group consisting of a pharmaceutically acceptable carrier(s) and auxiliary agent(s). 
     
     
         80 . The composition of  claim 74 , which further comprises optional components selected from the group consisting of a pharmaceutically acceptable carrier(s) and auxiliary agent(s). 
     
     
         81 . The recombinant engineered enzyme of  claim 29 , 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 41-45 and 125-128 in human trypsin I having the amino acid sequence shown in SEQ ID NO: 1.