Protease Variants

Abstract

The present invention relates to methods for producing variants of a parent RP-II pro-tease and the variants having altered properties as compared to the parent RP-II pro-tease.

1 - 14 . (canceled)  
     
     
         15 . A RP-II protease variant comprising at least one modification in an amino acid residue in a position located at a distance of 10 Å or less to the ion-binding site, preferably positions located at a distance of 6 Å or less.  
     
     
         16 . The variant of  claim 15 , wherein modifications are made in at least one of the positions: 1, 2, 3, 4, 5, 6, 7, 8, 143, 144, 145, 146, 158, 159, 160, 161, 162, 194, 199, 200, and 201, preferably positions 2, 3, 4, 5, 6, 7, 144, 159, 160, and 161, and especially the modifications D7E and D7Q in BLC (SEQ ID NO: 2), where the positions refer to BLC or corresponding positions.  
     
     
         17 . The variant of  claim 15 , wherein the modification comprises the substitution of a positively charged amino acid residue with a neutral or negatively charged residue, or the substitution of a neutral residue with a negatively charged residue or the deletion of a positively charged or neutral residue.  
     
     
         18 . The variant of  claim 15 , wherein the ion binding site is removed by modification in at least one of the positions corresponding to positions 144 and or 161 of BLC, especially the modifications H144R and/or D161R,K+H144Q,N in BLC (SEQ ID NO:2).  
     
     
         19 . A RP-II protease variant of  claim 15  comprising at least one modification in an amino acid residue in highly mobile regions in at least one of the positions corresponding to positions 26-31 (26, 27, 28, 29, 30, and 31); 89-91 (89, 90, and 91); 216-221 (216, 217, 218, 219, 220, and 221) of BLC.  
     
     
         20 . The variant of  claim 19 , wherein the parent is BLC and the modification comprises G30A and/or G91A.  
     
     
         21 . A RP-II protease variant of  claim 15  comprising at least one modification made in mobile regions in at least one of the positions corresponding to positions 51-56, (51, 52, 53, 54, 55, 56), 88-94, (88, 89, 90, 91, 92, 93, 94), 118-122 (118, 119, 120, 121, 122), and 173-183 (173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183) of BLC, preferably the regions 51-56 and 118-122.  
     
     
         22 . A RP-II protease variant of  claim 15  having at least one disulfide bridge provided by modifying the amino acid residues in positions 128 and 145 in BLC or corresponding positions to Cys, preferably the substitutions S145C and T128C in BLC or corresponding positions.  
     
     
         23 . A RP-II protease variant of  claim 15  having a modified surface charge distribution in comparison to the parent RP-II protease comprising modifications in at least one of the positions corresponding to positions 7, 17, 95, 109, 143, 174, 209, 216, of BLC, especially the modifications  
       D7N, S, T  
       Y17R, K, H  
       Y95R, K, H  
       T109R, K, H  
       Q143R, K, H  
       Q174R, K, H  
       E209Q, N  
       N216R, K, H  
       in BLC (SEQ ID NO. 1)  
     
     
         24 . A RP-II protease variant of  claim 15  exhibiting improved stability in comparison to the parent RP-II protease comprising at substitution to Pro in at least one of the positions corresponding to positions 18, 115, 185, 269 and 293 in BLC, especially one or more of the substitutions: T60P, S221P, G193P, V194P in BLC (SEQ ID NO. 1).  
     
     
         25 . A RP-II protease variant of  claim 15  comprising modifications in amino-acid residues in positions corresponding to positions 1, 8, 22-35 (22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35), 42-58 (42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58), 82-100 (82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100), 129-135 (129, 130, 131, 132, 133, 134, 135), 141-142, 153-156 (153, 154, 155, 156), 158, 161-171 (161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171), 188-193 (188, 189, 190, 191, 192, 193), 195, 201-207 (201, 202, 203, 204, 205, 206, 207), 210, 213-214, 217 in BLC at a distance of less than 10 Å from the active site residues.  
     
     
         26 . The RP-II protease variant of  claim 15 , further comprising at least one of the modifications (i) amino acid residues in positions that form part of an Asn-Gly sequence being modified by deletion or substitution, preferably with Asp, Gln, Ser, Pro, Thr, or Tyr; (ii) amino acid residues in positions that occupied by a Trp being modified by substitution with Phe, Thr, Gln or Gly; (iii) amino acid residues in positions that are occupied by Glu or Asp being modified by substitution with Ala; (iv) amino acid residues in positions that are in positions that are the 1 st  or 2 nd  position following a position occupied by a Glu or Asp residue being modified by substitution with a Pro; or (v) amino acid residues in positions that are occupied by a Met being modified by deletion or substitution, preferably with Ser or Ala.  
     
     
         27 . The RP-II protease of  claim 15  that is modified in a number of positions ranging from at least one and up to 50 positions, or from 1 to 45 positions, or from 1 to 40 positions, or from 1 to 35 positions, or from 1 to 30 positions, or from 1 to 25 positions, or from 1 to 20 positions, or from 1 to 15 positions, or from 1 to 14 positions, or from 1 to 13 positions, or from 1 to 12 positions, or from 1 to 11 positions, or from 1 to 10 positions, or from 1 to 9 positions, or from 1 to 8 positions, or from 1 to 7 positions, or from 1 to 6 positions, or from 1 to 5 positions, or from 1 to 4 positions, or from 1 to 3 positions, or from 1 to 2 positions, such modifications comprising substitutions, deletions, insertions and combinations thereof in the indicated number of positions.  
     
     
         28 . An isolated polynucleotide comprising a nucleic acid sequence, which encodes for a RP-II protease variant defined or produced in  claim 15 .  
     
     
         29 - 32 . (canceled)  
     
     
         33 . A detergent composition comprising a RP-II protease variant defined or produced in  claim 15 ,  
     
     
         34 - 37 . (canceled)