Cyclized cytokine and method for producing same

Abstract

The purpose of the present invention is to provide a method for producing a very stable, cyclized mutant protein such that high cyclization efficiency is achieved while the number of amino acids added is minimal and the biological properties of an original protein are maintained. In view of conformational information about the original protein, secondary structure-free regions at N/C terminal portions are deleted. Then, a protein database is screened for proteins with secondary structures similar to those of N/C terminal residues of a secondary structure-forming portion after the deletion. The screening results are used to determine the amino acid length of a loop structure through which the N-terminus and the C-terminus of the secondary structure-forming portion of the original protein are to be connected. A cyclized mutant protein is finally produced having a loop structure with the determined amino acid length.

We claim: 
     
         1 . A method for producing a cyclized mutant protein with biological properties not less than those of an original protein and with stability higher than that of the original protein by modifying, through the following steps (i) to (v), the original protein with a specific three-dimensional structure, the method comprising the steps of:
 (i) determining, based on conformational information about the original protein, secondary structure-free regions at an N-terminal portion and a C-terminal portion of the original protein and determining an N-terminus and a C-terminus of a secondary structure-forming portion of the original protein if the secondary structure-free regions are deleted;   (ii) screening a protein database for proteins with secondary structures similar to those of N-terminal residues and C-terminal residues of the secondary structure-forming portion;   (iii) determining, based on results of the screening, an amino acid length of a loop structure through which the N-terminus and the C-terminus of the secondary structure-forming portion of the original protein are to be connected;   (iv) designing and producing a linear mutant protein that can be subjected to cyclization so as to obtain a cyclized mutant protein in which the N-terminus and the C-terminus of the secondary structure-forming portion of the original protein are connected via the loop structure with the amino acid length determined; and   (v) linking an N-terminus and a C-terminus of the linear mutant protein by a chemical or biological method so that the cyclization is made to obtain the cyclized mutant protein.   
     
     
         2 . The method for producing a cyclized mutant protein according to  claim 1 , wherein the cyclization of step (v) uses a trans-splicing reaction mediated by split inteins. 
     
     
         3 . The method for producing a cyclized mutant protein according to  claim 1 , wherein the original protein with a specific three-dimensional structure is a cytokine with a helix bundle structure and the biological properties of the original protein involve affinity for a receptor. 
     
     
         4 . The method for producing a cyclized mutant protein according to  claim 3 , wherein the original protein with a specific three-dimensional structure is granulocyte colony-stimulating factor (G-CSF). 
     
     
         5 . (canceled) 
     
     
         6 . A cyclized mutant protein consisting of an amino acid sequence in which secondary structure-free regions at an N-terminal portion and a C-terminal portion of an original protein with a specific three-dimensional structure are deleted and a predetermined number of amino acid residues is added to each of an N-terminus and a C-terminus of a secondary structure-forming portion of the original protein after the deletion, wherein the predetermined number of amino acid residues is the number of amino acid residues, the number corresponding to the number of amino acids connecting secondary structures of N-terminal residues and C-terminal residues of a secondary structure-forming portion of another protein, the secondary structures being similar to those of the secondary structure-forming portion of the original protein. 
     
     
         7 . The cyclized mutant protein according to  claim 6 , wherein the original protein with a specific three-dimensional structure is a cytokine with a helix bundle structure. 
     
     
         8 . The cyclized mutant protein according to  claim 6 , wherein the original protein with a specific three-dimensional structure is a granulocyte colony-stimulating factor (G-CSF) consisting of an amino acid sequence set forth in SEQ ID NO: 1; 0 to 11 amino acid residues are deleted from an N-terminus of the amino acid sequence set forth in SEQ ID NO: 1; 0 to 3 amino acid residues are deleted from an C-terminus of the amino acid sequence; a serine or cysteine residue is added to an N-terminus and/or a C-terminus of the amino acid sequence after the deletion; and an amino acid residue other than proline is added to the C-terminus of the amino acid sequence after the deletion. 
     
     
         9 . The cyclized mutant protein according to  claim 6 , wherein the protein consists of a cyclized amino acid sequence set forth in any one of SEQ ID NOs: 6 to 9. 
     
     
         10 . A linear mutant protein designed so as to produce the cyclized mutant protein according to  claim 6 , wherein the linear mutant protein consists of an amino acid sequence in which secondary structure-free regions at an N-terminal portion and a C-terminal portion of an original protein with a specific three-dimensional structure are deleted and a predetermined number of amino acid residues is added to each of an N-terminus and a C-terminus of a secondary structure-forming portion of the original protein after the deletion; the predetermined number of amino acid residues is the number of amino acid residues, the number corresponding to the number of amino acids connecting secondary structures of N-terminal residues and C-terminal residues of a secondary structure-forming portion of another protein, the secondary structures being similar to those of the secondary structure-forming portion of the original protein; and a most N-terminus amino acid residue and a most C-terminus amino acid residue of the predetermined number of amino acid residues added are amino acid residues that enable the linear mutant protein to be subject to cyclization. 
     
     
         11 . The linear mutant protein according to  claim 10 , wherein the protein is designed so as to produce the cyclized mutant protein that consists of a cyclized amino acid sequence set forth in any one of SEQ ID NOs: 6 to 9 and consists of an amino acid sequence set forth in any one of SEQ ID NOs: 2 to 5. 
     
     
         12 . The linear mutant protein according to  claim 10 , wherein the protein is designed so as to produce the cyclized mutant protein that consists of a cyclized amino acid sequence set forth in any one of SEQ ID NOs: 6 to 9 by using, as split inteins, DnaE-C (C-intein) and DnaE-N (N-intein) of DnaE derived from  Nostoc punctiforme  and consists of an amino acid sequence set forth in any one of SEQ ID NOs: 10 to 13. 
     
     
         13 . A nucleic acid comprising a nucleotide sequence encoding an amino acid sequence of the mutant protein according to  claim 6 . 
     
     
         14 . A recombinant vector comprising the nucleic acid according to  claim 13 . 
     
     
         15 . A transformant comprising the recombinant vector according to  claim 14 . 
     
     
         16 . A method for producing the mutant protein according to  claim 6 , comprising using a transformant, said transformant comprising a recombinant vector, said recombinant vector comprising a nucleic acid, said nucleic acid comprising a nucleotide sequence encoding an amino acid sequence of the mutant protein according to  claim 6 . 
     
     
         17 . A pharmaceutical composition comprising, as an active ingredient, the mutant protein according to  claim 6 . 
     
     
         18 . A pharmaceutical composition for treatment of neutropenia, comprising, as an active ingredient, the mutant protein according to  claim 6 . 
     
     
         19 . The cyclized mutant protein according to  claim 7 , wherein the cytokine is selected from granulocyte colony-stimulating factor, erythropoietin, and interferon α. 
     
     
         20 . A method for producing the cyclized mutant protein of  claim 6 , comprising the steps of:
 (i) determining, based on conformational information about the original protein, secondary structure-free regions at an N-terminal portion and a C-terminal portion of the original protein and determining an N-terminus and a C-terminus of a secondary structure-forming portion of the original protein if the secondary structure-free regions are deleted;   (ii) screening a protein database for proteins with secondary structures similar to those of N-terminal residues and C-terminal residues of the secondary structure-forming portion;   (iii) determining, based on results of the screening, an amino acid length of a loop structure through which the N-terminus and the C-terminus of the secondary structure-forming portion of the original protein are to be connected;   (iv) designing and producing a linear mutant protein that can be subjected to cyclization so as to obtain a cyclized mutant protein in which the N-terminus and the C-terminus of the secondary structure-forming portion of the original protein are connected via the loop structure with the amino acid length determined; and   (v) linking an N-terminus and a C-terminus of the linear mutant protein by a chemical or biological method so that the cyclization is made to obtain the cyclized mutant protein,   to thereby produce the cyclized mutant protein of  claim 6 .