Ob-fold used as scaffold for engineering new specific binders

Abstract

The present invention pertains to the field of protein engineering, and provides means for obtaining stable molecules that specifically bind to a target selected amongst a large variety of ligands families. In particular, the present invention provides methods for obtaining a molecule specifically binding to a target of interest, through a combinatorial mutation/selection approach with an OB-fold protein as a starting molecule. In particular, the target of interest can be of a different chemical nature form that of the native target of the OB-fold protein used as the starting molecule.

1 - 3 . (canceled) 
     
     
         4 . A combinatorial library corresponding to the randomization of 5 to 32 of the binding interface of an OB-fold protein with its native ligand. 
     
     
         5 . The combinatorial library of  claim 4 , further comprising an insertion of 1 to 15 random amino acid residues in loop 3, an insertion of 1 to 15 random amino acid residues in loop 4, an insertion of 1 to 20 random amino acid residues in loop 1, or a combination thereof. 
     
     
         6 . The combinatorial library of  claim 4 , wherein the OB-fold protein is Sac7d or Sac7e from  Sulfolobus acidocaldarius , or Sso7d, from  Sulfolobus solfataricus , or DBP 7 from  Sulfolobus tokodaii , or Ssh7b from  Sulfolobus shibatae , or Ssh7a from  Sulfolobus shibatae , or p7ss from  Sulfolobus solfataricus.    
     
     
         7 . The combinatorial library of  claim 6 , which corresponds to the randomization of residues of Sac7d selected from the group consisting of V2, K3, K5, K7, Y8, K9, G10, E14, T17, K21, K22, W24, V26, G27, K28, M29, S31, T33, D36, N37, G38, K39, T40, R42, A44, S46, E47, K48, D49, A50 and P51. 
     
     
         8 . The combinatorial library of  claim 7 , which corresponds to the randomization of 11, 12, 13, 14, 15, 16, 17 or 18 residues of Sac7d selected from the group consisting of K7, Y8, K9, K21, K22, W24, V26, K28, M29, S31, T33, K39, T40, R42, A44, S46, E47 and K48. 
     
     
         9 . The combinatorial library of  claim 8 , which corresponds to the randomization of 11, 12, 13, 14, 15 or 16 residues of Sac7d selected from the group consisting of K7, Y8, K9, K21, K22, W24, V26, K28, M29, S31, T33, K39, T40, R42, A44 and S46. 
     
     
         10 . The combinatorial library of  claim 9 , in which the randomized residues comprise at least the residues K7, Y8, K9, W24, V26, M29, S31, T33, R42, A44 and
 S46 of Sac7d.   
     
     
         11 . The combinatorial library of  claim 10 , which corresponds to the randomization of the residues K7, Y8, K9, W24, V26, M29, S31, T33, R42, A44 and S46 of Sac7d. 
     
     
         12 . The combinatorial library of  claim 10 , in which one, two or three additional residues of Sac7d selected amongst from the group consisting of K21, K22 and T40 are also randomized. 
     
     
         13 . The combinatorial library of  claim 12 , which corresponds to the randomization of the residues K7, Y8, K9, K21, K22, W24, V26, M29, S31, T33, R42, A44 and S46 of Sac7d. 
     
     
         14 . The combinatorial library of  claim 12 , which corresponds to the randomization of the residues K7, Y8, K9, K21, K22, W24, V26, M29, S31, T33, T40, R42, A44 and S46 of Sac7d. 
     
     
         15 . The combinatorial library of  claim 6 , wherein 1 to 15 random amino acid residues are inserted in the region corresponding to the amino acids in position 25 to 30 of Sac7d, to 15 random amino acid residues are inserted in the region corresponding to the amino acids in position 35 to 40 of Sac7d, 1 to 20 random amino acid residues are inserted in the region corresponding to the amino acids in position 7 to 12 of Sac7d, or a combination thereof. 
     
     
         16 . The combinatorial library of  claim 6 , wherein 1 to 4 amino acid residues selected from the group consisting of A59, R60, A61 and E64 are deleted. 
     
     
         17 . (canceled) 
     
     
         18 . A process for engineering a molecule specifically binding to a target, comprising selecting, in a combinational library according to  claim 4 , variants of said OB-fold protein which bind to said target. 
     
     
         19 . The process of  claim 18 , wherein said target is a peptide, a protein, an oligosaccharide, a single-stranded DNA, a double-stranded DNA, an RNA, a metallic ion, or a carbohydrate. 
     
     
         20 . The process of  claim 18 , wherein said selecting is performed by ribosome display. 
     
     
         21 . The process of  claim 20 , in which 2, 3, 4 or 5 rounds of selection are performed by ribosome display. 
     
     
         22 . The process of any of  claim 20 , further comprising isolating and characterizing a molecule specifically binding to said target. 
     
     
         23 . The process of  claim 22 , wherein isolating and characterizing comprises:
 (i) mRNAs from a pool selected by ribosome display are reversed-transcribed and amplified by PCR, and the resulting DNAs are cloned into expression vectors;   (ii) bacteria are transformed by said expression vectors and individual clones are grown; and   (iii) proteins extracted from said bacterial clones are tested for their binding to the target.   
     
     
         24 . The process  claim 22 , further comprising making a fusion protein comprising, as a first moiety, the isolated and characterized molecule, and at least a second moiety. 
     
     
         25 . The process of  claim 24 , wherein said at least second moiety is selected from the group consisting of enzymes, marker proteins, therapeutic proteins, and binders with different specificities than said first moiety. 
     
     
         26 . A protein obtained through the process according to  claim 18 , wherein said protein specifically binds to PulD, and wherein the sequence thereof is selected from the group consisting of SEQ ID Nos: 2, 3, 4, 5, 6, 7 and 8. 
     
     
         27 . A protein obtained through the process according to  claim 18 , wherein said protein specifically binds to GarA, and wherein the sequence thereof is SEQ ID No: 47. 
     
     
         28 . The combinatorial library according to  claim 4 , corresponding to the randomization of 8 to 20 of the binding interface of an OB-fold protein with its native ligand. 
     
     
         29 . The combinatorial library according to  claim 4 , corresponding to the randomization of 11 to 16 of the binding interface of an OB-fold protein with its native ligand. 
     
     
         30 . The combinatorial library of  claim 6 , wherein 1 to 15 random amino acid residues are inserted between G27 and K28 of Sac7d, 1 to 15 random amino acid residues are inserted between N37 and G38 of Sac7d, 1 to 20 random amino acid residues are inserted between K9 and G10 of Sac7d, or a combination thereof.