Related ribonucleases in which an amino acid sequence of a known ribonuclease is preceded by at least one residue at the n-terminus, and methods of making them recombinantly

Abstract

New RNases, and methods for making them, are disclosed. Each includes an amino acid sequence that is disclosed in U.S. Pat. No. 6,239,257 B1, but that is preceded by a different N-terminus residue or leader sequence.

1 . A vector containing DNA that encodes the Ribonuclease of SEQ ID NO:1. 
     
     
         2 . The vector of  claim 1 , wherein the vector is pET11d plasmid. 
     
     
         3 . The vector of  claim 1 , wherein the vector is pET22b plasmid. 
     
     
         4 . A vector containing DNA that encodes the Ribonuclease of SEQ ID NO:17. 
     
     
         5 . A vector containing DNA that encodes the Ribonuclease of SEQ ID NO:34. 
     
     
         6 . A vector containing DNA that encodes the Ribonuclease of SEQ ID NO:51. 
     
     
         7 . A vector containing DNA that encodes the Ribonuclease of SEQ ID NO:55. 
     
     
         8 . A protein having the amino acid sequence of SEQ ID NO:59 produced recombinantly. 
     
     
         9 . A protein produced recombinantly and having the amino acid sequence of SEQ ID NO:1 after its initial methionine residue has been cleaved off. 
     
     
         10 . A protein having the amino acid sequence of SEQ ID NO:60 produced recombinantly. 
     
     
         11 . A protein produced recombinantly and having the amino acid sequence of SEQ ID NO:17 after its initial methionine residue has been cleaved off. 
     
     
         12 . A protein having the amino acid sequence of SEQ ID NO:61 produced recombinantly. 
     
     
         13 . A protein produced recombinantly and having the amino acid sequence of SEQ ID NO:34 after its initial methionine residue has been cleaved off. 
     
     
         14 . A protein having the amino acid sequence of SEQ ID NO:68 produced recombinantly. 
     
     
         15 . A protein produced recombinantly and having the amino acid sequence of SEQ ID NO:51 after its initial methionine residue has been cleaved off. 
     
     
         16 . A protein having the amino acid sequence of SEQ ID NO:59. 
     
     
         17 . A conservatively modified variant of the protein of  claim 16 . 
     
     
         18 . A gene that when expressed in a host encodes the protein of  claim 16 . 
     
     
         19 . A gene that when expressed in a host encodes the protein of  claim 17 . 
     
     
         20 . A protein having the amino acid sequence of SEQ ID NO:60. 
     
     
         21 . A conservatively modified variant of the protein of  claim 20 . 
     
     
         22 . A gene that when expressed in a host encodes the protein of  claim 20 . 
     
     
         23 . A gene that when expressed in a host encodes the protein of  claim 21 . 
     
     
         24 . A protein having the amino acid sequence of SEQ ID NO:61. 
     
     
         25 . A conservatively modified variant of the protein of  claim 24 . 
     
     
         26 . A gene that when expressed in a host encodes the protein of  claim 24 . 
     
     
         27 . A gene that when expressed in a host encodes the protein of  claim 25 . 
     
     
         28 . A protein having the amino acid sequence of SEQ ID NO:63. 
     
     
         29 . A protein having the amino acid sequence of SEQ ID NO:65. 
     
     
         30 . A protein having the amino acid sequence of SEQ ID NO:67. 
     
     
         31 . A protein having the amino acid sequence of SEQ ID NO:1 preceded at its N-terminal end by a leader sequence that has at least the first and at most all of the residues of SEQ ID NO:64 in order. 
     
     
         32 . A protein having the amino acid sequence of SEQ ID NO:1 preceded at its N-terminal end by a leader sequence that has at least the first and at most all of the residues of SEQ ID NO:66 in order. 
     
     
         33 . An isolated protein having the amino acid sequence of SEQ ID NO:68. 
     
     
         34 . A conservatively modified variant of the protein of  claim 33 . 
     
     
         35 . A gene that when expressed in a host encodes the protein of  claim 33 . 
     
     
         36 . A gene that when expressed in a host encodes the protein of  claim 34 . 
     
     
         37 . A protein having the amino acid sequence of SEQ ID NO:69. 
     
     
         38 . A conservatively modified variant of the protein of  claim 37 . 
     
     
         39 . A gene that when expressed in a host encodes the protein of  claim 37 . 
     
     
         40 . A gene that when expressed in a host encodes the protein of  claim 38 . 
     
     
         41 . A conjugate protein comprising the protein of  claim 37  and a targeting moiety conjugated to the cysteine residue at position 71. 
     
     
         42 . A protein having the amino acid sequence of SEQ ID NO:17 preceded at its N-terminal end by a leader sequence that has at least the first and at most all of the residues of SEQ ID NO:64 in order. 
     
     
         43 . A protein having the amino acid sequence of SEQ ID NO:17 preceded at its N-terminal end by a leader sequence that has at least the first and at most all of the residues of SEQ ID NO:66 in order. 
     
     
         44 . A protein having the amino acid sequence of SEQ ID NO:34 preceded at its N-terminal end by a leader sequence that has at least the first and at most all of the residues of SEQ ID NO:64 in order. 
     
     
         45 . A protein having the amino acid sequence of SEQ ID NO:34 preceded at its N-terminal end by a leader sequence that has at least the first and at most all of the residues of SEQ ID NO:66 in order. 
     
     
         46 . An isolated protein having the amino acid sequence of SEQ ID NO:51 preceded at its N-terminal end by a leader sequence, the leader sequence having a) the first residue of SEQ ID NO:64, or b) all residues of SEQ ID NO:64, or c) any fragment of SEQ ID NO:64 that comprises the first residue of SEQ ID NO:64, said isolated protein being active against A-253 cells. 
     
     
         47 . An isolated protein having the amino acid sequence of SEQ ID NO:51 preceded at its N-terminal end by a leader sequence having a) the first residue of SEQ ID NO:66, or b) all residues of SEQ ID NO:66, or c) any fragment of SEQ ID NO:66 that comprises the first residue of SEQ ID NO:66, said isolated protein being active against A-253 cells. 
     
     
         48 . A protein having the amino acid sequence of SEQ ID NO:55. 
     
     
         49 . A fusion protein having the amino acid sequence of SEQ ID NO:70. 
     
     
         50 . A conservatively modified variant of the protein of  claim 49 . 
     
     
         51 . A gene that when expressed in a host encodes the protein of  claim 49 . 
     
     
         52 . A gene that when expressed in a host encodes the protein of  claim 50 . 
     
     
         53 . A fusion protein comprising a) the protein of SEQ ID NO:1 or a conservatively modified variant thereof and b) a targeting moiety. 
     
     
         54 . The fusion protein of  claim 57 , further comprising a linker sequence linking the protein and the targeting moiety. 
     
     
         55 . A conjugated fusion protein comprising a) the fusion protein of SEQ ID NO:70 or a conservatively modified variant thereof and b) a targeting moiety conjugated to the cysteine residue located at position 71. 
     
     
         56 . A vector containing DNA of SEQ ID NO:71 encoding the Ribonuclease of SEQ ID NO:70. 
     
     
         57 . An isolated protein having the amino acid sequence of SEQ ID NO:51 preceded at its N-terminal end by a leader sequence, the leader sequence having at least the first and at most all of the residues of SEQ ID NO:62 in order, said isolated protein being active against A-253 cells.