Fusion protein and use thereof

Abstract

The invention relates to a fusion protein and a method for the generation of the fusion protein of the invention. Further, the invention relates to the use of the fusion protein of the invention for the generation of induced pluripotent cells. Moreover, the invention relates to a composition comprising at least one fusion protein of the invention.

1 . A Fusion protein comprising:
 a) a protein transduction domain (PTD) comprising 6 to 12 basic amino acids; and   b) at least one transcription factor.   
     
     
         2 . The fusion protein of  claim 1 , wherein the PTD further comprises hydrophobic amino acids. 
     
     
         3 . The fusion protein of  claim 1 , wherein the PTD further comprises at least one of TAT, Penetratin, HSV-VP22, Transportan, K-FGF, Oligoarginine, Arg-9, and peptides consisting of combinations of Arginine and Lysine residues. 
     
     
         4 . The fusion protein of  claim 1 , wherein the PTD is comprised of no more than 20 amino acids. 
     
     
         5 . The fusion protein of  claim 1 , wherein the transcription factor is selected from the group consisting of: stem cell factors, such as Oct4, Sox2, nanog, klf4, lin28, hTERT, myc, SV40; differentiation factors, such as hox-genes, pax genes; reprogramming factors, such as HMGB, Msx1; tumor suppressors, such as p53, Pten; and anti-apoptose factors, such as bcl-2. 
     
     
         6 . The fusion protein of  claim 5 , wherein the transcription factor is one of Sox2 and Oct4. 
     
     
         7 . The fusion protein of  claim 1 , further comprising a nuclear localization signal (NLS). 
     
     
         8 . The fusion protein of  claim 1 , wherein the fusion protein comprises an artificial trans activation domain (ATAD). 
     
     
         9 . The fusion protein of  claim 1 , further comprising a linker selected from the group consisting of Proline, Glycine, Alanine, Leucine, Glutamic acid, Threonine, and Serine. 
     
     
         10 . The fusion protein of  claim 1 , further comprising at least one end group selected from the group consisting of Alanine and Glycine. 
     
     
         11 . The fusion protein of  claim 1 , said fusion protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8. 
     
     
         12 . A Fusion protein comprising:
 a) at least one transcription factor, wherein from 6 to 12 amino acids of said transcription factor have been replaced by basic amino acids.   
     
     
         13 . The fusion protein of  claim 12 , wherein the transcription factor is selected from the group consisting of: stem cell factors, such as Oct4, Sox2, nanog, klf4, 1 in28, hTERT, myc, SV40; differentiation factors, such as hox-genes, pax genes; reprogramming factors, such as HMGB, Msx1; tumor suppressors, such as p53, Pten; and anti-apoptose factors, such as bcl-2. 
     
     
         14 . The fusion protein of  claim 13 , wherein the transcription factor is one of Sox2 and Oct4. 
     
     
         15 . The fusion protein of  claim 12 , further comprising a nuclear localization signal (NLS). 
     
     
         16 . The fusion protein of  claim 12 , wherein the fusion protein comprises an artificial transactivation domain (ATAD). 
     
     
         17 . The fusion protein of  claim 12 , further comprising a linker selected from the group consisting of Proline, Glycine, Alanine, Leucine, Glutamic acid, Threonine, and Serine. 
     
     
         18 . The fusion protein of  claim 12 , further comprising at least one end group selected from the group consisting of Alanine and Glycine. 
     
     
         19 . A method of utilizing the fusion protein of  claim 1  for the generation of induced pluripotent cells or for the differentiation of pluripotent cells. 
     
     
         20 . A composition comprising the fusion protein of  claim 1  and at least one stabilizer. 
     
     
         21 . The composition of  claim 21 , wherein the stabilizer is a lipid rich serum. 
     
     
         22 . A method of generating the fusion protein of  claim 1 , the method comprising the steps of:
 a. constructing a DNA sequence comprising a sequence encoding a PTD and a sequence encoding a transduction factor;   b. cloning the DNA sequence into a suitable vector;   c. transforming the vector in a suitable expression system; and   d. optionally purifying the expressed fusion proteins.   
     
     
         23 . The method of  claim 22 , wherein the vector is a plasmid vector. 
     
     
         24 . The method of  claim 23 , wherein the vector is based on a pTriEx expression plasmid. 
     
     
         25 . The method of  claim 22 , wherein the expression system is selected from the group consisting of eukaryotic expression systems, bacterial expression systems, insect cell expression systems, and mammalian expression systems. 
     
     
         26 . The method of  claim 25 , wherein the bacterial expression system is an  Escherichia coli  expression strain. 
     
     
         27 . The method of  claim 22 , wherein the expression system is a cell-free expression system. 
     
     
         28 . The method of  claim 22 , wherein the method further comprises purifying expressed fusion proteins as step d). 
     
     
         29 . A nucleic acid comprising a nucleotide sequence encoding the fusion protein of  claim 1 . 
     
     
         30 . A method for generating induced pluripotent cells, the method comprising the step of:
 a. contacting at least one fusion protein as defined in  claim 1  with at least one target cell.   
     
     
         31 . A method for generating induced pluripotent cells, the method comprising the steps of:
 a. carrying out steps a) to c) of  claim 22 , wherein step c) is carried out in a cell-free expression system; and   b. contacting the cell-free expression system of step a) with at least one target cell.   
     
     
         32 . The method of  claim 30 , wherein the step of contacting is carried out for a duration of from 6 hours to 60 days. 
     
     
         33 . The method of  claim 30 , wherein the at least one target cell is selected from the group consisting of embryonic stem cells, adult stem cells, and somatic cells of mammals, respectively, such as cells of flies, such as  D. melanogaster , worms, such as  C. elegans  as well as lower eukaryotes, such as yeasts, such as  S. cerevisiae.    
     
     
         34 . The fusion protein of  claim 5 , wherein the stem cell factor is selected from the group comprising Oct4, Sox2, nanog, klf4, 1 in28, hTERT, myc, and SV40; the differentiation factor is selected from the group comprising hox-genes and pax genes; the reprogramming factor is selected from the group comprising HMGB and Msx1; the tumor suppressor is selected from the group comprising p53 and Pten; and the antiapoptose factor is bcl-2. 
     
     
         35 . The fusion protein of  claim 13 , wherein the stem cell factor is selected from the group comprising Oct4, Sox2, nanog, klf4, 1 in28, hTERT, myc, and SV40; the differentiation factor is selected from the group comprising hox-genes and pax genes; the reprogramming factor is selected from the group comprising HMGB and Msx1; the tumor suppressor is selected from the group comprising p53 and Pten; and the antiapoptose factor is bcl-2. 
     
     
         36 . The method of  claim 33 , wherein the cells are derived from at least one of the group consisting of cells of flies, worms, and lower eukaryotes. 
     
     
         37 . The method of  claim 36 , wherein the flies are  D. melanogaster , the worms are  C. elegans , and the lower eukaryotes are yeasts. 
     
     
         38 . The method of  claim 37 , wherein the yeasts are  S. cerevisiae.