Fusion Proteins With Cleavable Spacers and Uses Thereof

Abstract

A polypeptide comprising a first protein domain, a second protein domain, and a dithiocyclopeptide spacer containing at least one protease cleavage site, wherein the dithiocyclopeptide is exogenous relative to the first or second protein domain, and wherein the first and second protein domains are operably linked by the dithiocyclopeptide. Also disclosed are methods of producing the polypeptide and delivering the protein domains into a cell.

1 . A polypeptide comprising:
 a first protein domain;   a second protein domain; and   a dithiocyclopeptide spacer containing at least one protease cleavage site, wherein the dithiocyclopeptide is exogenous relative to the first or second protein domain, and wherein the first and second protein domains are operably linked by the dithiocyclopeptide.   
     
     
         2 . The polypeptide of  claim 1 , wherein the dithiocyclopeptide is cyclized by a disulfide bond. 
     
     
         3 . The polypeptide of  claim 2 , wherein the dithiocyclopeptide is cleaved by the protease at the protease cleavage site. 
     
     
         4 . The polypeptide of  claim 1 , wherein the dithiocyclopeptide is cleaved by the protease at the protease cleavage site. 
     
     
         5 . The polypeptide of  claim 1 , wherein the first protein domain is a granulocyte-colony stimulating factor (G-CSF) domain. 
     
     
         6 . The polypeptide of  claim 5 , wherein the second protein domain is a transferrin (Tf) domain. 
     
     
         7 . The polypeptide of  claim 1 , wherein the second protein domain is a Tf domain. 
     
     
         8 . The polypeptide of  claim 1 , wherein the dithiocyclopeptide contains a thrombin or trypsin cleavage site. 
     
     
         9 . The polypeptide of  claim 6 , wherein the dithiocyclopeptide comprises at least one of LEAGCKNFFPR (SEQ ID NO: 3) and SFTSCGSLE (SEQ ID NO: 4). 
     
     
         10 . The polypeptide of  claim 1 , wherein the polypeptide is a recombinant polypeptide. 
     
     
         11 . A nucleic acid comprising a DNA sequence encoding the polypeptide of  claim 10 . 
     
     
         12 . A cell comprising the nucleic acid of  claim 11 . 
     
     
         13 . A method of producing a polypeptide, comprising cultivating the cell of  claim 12  under conditions that allow expression of the polypeptide. 
     
     
         14 . The method of  claim 13 , further comprising collecting the polynucleotide. 
     
     
         15 . The method of  claim 14 , further comprising cleaving the polypeptide with the protease. 
     
     
         16 . A method of delivering protein domains into a cell, comprising contacting a cell with the polypeptide of  claim 3  under conditions that allow transport of the polypeptide into the cell, wherein the disulfide bond in the dithiocyclopeptide is reduced during the transport or within the cell, thereby separating the first protein domain from the second protein domain. 
     
     
         17 . The method of  claim 16 , wherein the first protein domain is a G-CSF domain. 
     
     
         18 . The method of  claim 17 , wherein the second protein domain is a Tf domain. 
     
     
         19 . The method of  claim 18 , wherein the cell expresses transferrin receptor (TfR). 
     
     
         20 . The method of  claim 16 , wherein the second protein domain is a Tf domain. 
     
     
         21 . The method of  claim 20 , wherein the cell expresses TfR.