Method for refining protein including self-cutting cassette and use thereof

Abstract

The present invention relates to a self-cleaving fusion protein including a target protein, a peptide consisting of amino acid sequence represented by LPXTG, a domain of Sortase A having cleaving function, and a tag, which are sequentially positioned from the amino terminal; a nucleic acid encoding the same; an expression vector including the nucleic acid of the present invention; and a cell transformed with the expression vector of the present invention. In addition, the present invention relates to a method for refining a target protein including culturing, dissolving, and purifying the transformed cell, and a method for preparing a therapeutic antibody-drug conjugate by using the purifying method.

1 . A self-cleaving fusion protein comprising:
 (i) a target protein;   (ii) a peptide represented by Formula I below;   (iii) a domain of Sortase A having cleaving function;   (iv) a tag, wherein (i) to (iv) are sequentially positioned from amino terminus to a carboxyl terminus of the fusion protein,
   L-P-X-T-G (SEQ ID NO: 58),  [Formula I]
 
   wherein L represents Leucine, P represents Proline, X represents an any amino acid, T represents Threonine, and G represents Glycine.   
     
     
         2 . The self-cleaving fusion protein according to  claim 1 , further comprising a peptide linker between a peptide represented by Formula I and a domain of Sortase A having cleaving function. 
     
     
         3 . The self-cleaving fusion protein according to  claim 1 , wherein X in Formula I is glutamic acid. 
     
     
         4 . The self-cleaving fusion protein according to  claim 2 , wherein the peptide linker is selected from the group consisting of a natural linker, a flexible linker, a helical linker, a positively charged linker, a negatively charged linker and a coiled coil linker. 
     
     
         5 . The self-cleaving fusion protein according to  claim 2 , wherein the peptide linker is represented by Sc(SG4)l(GGSSRSS)GdSe (SEQ ID NO: 4), in which S represents Serine, G represents Glycine, R represents Arginine, c represents 0 to 5, d represents 0 to 5, e represents 0 to 5, and l represents 0 to 10. 
     
     
         6 . The self-cleaving fusion protein according to  claim 2 , wherein the peptide linker consists of 19 to 40 amino acids. 
     
     
         7 . The self-cleaving fusion protein according to  claim 2 , wherein the peptide linker consists of 19 to 25 amino acids. 
     
     
         8 . The self-cleaving fusion protein according to  claim 2 , wherein the peptide linker comprises an amino acid sequence represented by SEQ ID NO: 7. 
     
     
         9 . The self-cleaving fusion protein according to  claim 1 , wherein the Sortase A is derived from  Staphylococcus aureus  ( S. aureus ). 
     
     
         10 . The self-cleaving fusion protein according to  claim 1 , wherein the domain of Sortase A having cleaving function comprises an amino acid sequence represented by SEQ ID NO: 8. 
     
     
         11 . The self-cleaving fusion protein according to  claim 1 , wherein the tag is selected from the group consisting of a poly-histidine tag, a glutathione-S-transferase tag, a Hemagglutinin tag, a FLAG tag, a Myc tag, a maltose binding protein tag, a chitin binding protein tag, and a fluorescent tag. 
     
     
         12 . The self-cleaving fusion protein according to  claim 11 , wherein the tag is a poly-histidine tag. 
     
     
         13 . The self-cleaving fusion protein according to  claim 12 , wherein the poly-histidine tag comprises 6 to 12 sequential histidines. 
     
     
         14 . The self-cleaving fusion protein according to  claim 1 , wherein the target protein is selected from the group consisting of polymer proteins, glycoproteins, cytokines, growth factors, blood preparations, vaccines, hormones, enzymes and antibodies. 
     
     
         15 . The self-cleaving fusion protein according to  claim 1 , wherein the target protein is a portion or whole of a light chain or a heavy chain of an antibody. 
     
     
         16 . The self-cleaving fusion protein according to  claim 15 , wherein the target protein is a light chain variable region (VL) or a heavy chain variable region (VH) of an antibody. 
     
     
         17 . The self-cleaving fusion protein according to  claim 1 , wherein the fusion protein comprises an amino acid sequence represented by SEQ ID NO: 17 or 18. 
     
     
         18 . A nucleic acid encoding the self-cleaving fusion protein according to  claim 1 . 
     
     
         19 . An expression vector comprising the nucleic acid of  claim 18 . 
     
     
         20 . A host cell transformed with the expression vector of  claim 19 . 
     
     
         21 . The host cell according to  claim 20 , wherein the host cell is a prokaryotic or eukaryotic cell. 
     
     
         22 . The host cell according to  claim 21 , wherein the host cell is  Escherichia coli.    
     
     
         23 . (canceled) 
     
     
         24 . A method for purifying a target protein comprising: (1) culturing cells of  claim 20  to obtain cell lysates; and (2) purifying the target protein from the cell lysates. 
     
     
         25 . The method for purifying a target protein of  claim 24 , wherein step (2) comprises:
 (a) injecting the cell lysates into a column bound to a tag in a fusion protein;   (b) washing the column;   (c) equilibrating the column by using a cleavage buffer including at least one selected from the group consisting of calcium and triglycine to perform a cleaving reaction; and   (d) obtaining the cleavage buffer from the column to obtain the target protein from which the tag is removed.   
     
     
         26 .- 28 . (canceled) 
     
     
         29 . A method of preparing a therapeutic antibody-drug conjugate comprising:
 (1) reacting the self-cleaving fusion protein of  claim 1  with a triglycine-drug (GGG-drug) in a cleavage buffer including calcium to conjugate the triglycine-drug (GGG-drug) to the target protein; and   (2) recovering a conjugate of the target protein in which the tag has been replaced with the triglycine-drug.   
     
     
         30 . The method of preparing a therapeutic antibody-drug conjugate of  claim 29 , wherein the cleavage buffer in step (1) comprises 0.1 to 10 mM of calcium. 
     
     
         31 .- 32 . (canceled) 
     
     
         33 . The method of preparing a therapeutic antibody-drug conjugate of  claim 29 , wherein the target protein is an antibody against a tumor surface antigen.