Destabilising domains for conditionally stabilising a protein

Abstract

The present disclosure relates to mutant polypeptides derived from Escherichia coli dihydrofolate reductase (DHFR) which can be fused to a polypeptide of interest for efficient conditional modulation of its activity. Also disclosed are polynucleotides encoding such mutant polypeptides, vectors comprising such polynucleotides, and the use of such polypeptides, polynucleotides and vectors for treating a disorder.

1 . A mutant polypeptide derived from DHFR comprising or consisting of a sequence differing from SEQ ID NO: 1 and SEQ ID NO: 2 in at least one of the following positions: R12, N18, M42, Y100, D122, P126, D127, W133 and/or F153, said sequence having at least 70% identity, such as at least 75% identity, such as 80% identity, such as at least 85% identity, such as at least 90% identity, such as at least 95% identity, such as at least 96% identity, such as at least 97% identity, such as at least 98% identity, such as at least 99% identity to SEQ ID NO: 1 or SEQ ID NO: 2, with the proviso that the mutant polypeptide does not differ from SEQ ID NO: 1 and SEQ ID NO: 2 only by a Y100I mutation. 
     
     
         2 . The polypeptide of  claim 1 , wherein the sequence of the polypeptide differs from SEQ ID NO: 1 and SEQ ID NO: 2 in at least one of the following positions: W133 and F153 
     
     
         3 . The polypeptide of any one of the preceding claims, wherein the mutant further comprises at least one mutation at position Y100, such as Y100E or Y100I, preferably Y100E. 
     
     
         4 . The polypeptide of any one of the preceding claims, wherein the sequence differs from SEQ ID NO: 1 by the presence of a mutation which at least partly removes the side chain at the mutated position. 
     
     
         5 . The polypeptide of any one of the preceding claims, wherein the mutation which at least partly removes the side chain is a mutation to a glycine G or to an alanine A in at least one of positions R12, N18, M42, P126, D127, W133 and/or F153. 
     
     
         6 . The polypeptide of any one of the preceding claims, wherein the mutant comprises one or more of the mutations selected from the group consisting of: W133A, F153G, F153D, R12A, M42A, D122A, P126Y, P126D, P126R, F153A, M42A Y100E, R12A Y100E, N18T Y100I and D127N Y100I. 
     
     
         7 . The polypeptide of any one of the preceding claims, wherein the mutant further comprises a mutation in the TMP binding pocket of SEQ ID NO: 1. 
     
     
         8 . The polypeptide of any one of the preceding claims, wherein the polypeptide does not have a G67S mutation. 
     
     
         9 . The polypeptide of any one of  claims 1  to  2 , wherein the polypeptide does not have a Y100 mutation. 
     
     
         10 . The polypeptide of any one of the preceding claims, wherein the sequence differs from SEQ ID NO: 1 or SEQ ID NO: 2 at least in positions 150 and W133, or at least in positions R52 and W133, or at least in positions L54 and W133, or at least in positions P55 and W133, preferably the sequence differs from SEQ ID NO: 1 or SEQ ID NO: 2 in positions 150 and W133, or in positions R52 and W133, or in positions L54 and W133, or in positions P55 and W133. 
     
     
         11 . The polypeptide of any one of the preceding claims, wherein the sequence differs from SEQ ID NO: 1 or SEQ ID NO: 2 at least in positions 150 and F153, or at least in positions R52 and F153, or at least in positions L54 and F153, or at least in positions P55 and F153, preferably the sequence differs from SEQ ID NO: 1 or SEQ ID NO: 2 in positions 150 and F153, or in positions R52 and F153, or in positions L54 and F153, or in positions P55 and F153. 
     
     
         12 . The polypeptide of any one of the preceding claims, wherein the mutant differs from SEQ ID NO: 1 and SEQ ID NO: 2 in at least one further position, such as at least two further positions, such as at least three further positions. 
     
     
         13 . The polypeptide of any one of the preceding claims, wherein the further position is I50, R52, L54 or P55 of SEQ ID NO: 1 or SEQ ID NO: 2. 
     
     
         14 . The polypeptide of any one of the preceding claims, wherein the mutant polypeptide is a W133A mutant of SEQ ID NO: 1, an F153G mutant of SEQ ID NO: 1, an F153D mutant of SEQ ID NO: 1, an R12A Y100E mutant of SEQ ID NO: 1, an N18T Y100E mutant of SEQ ID NO: 1, an M42A Y100E mutant of SEQ ID NO: 1, a Y100I D127N mutant of SEQ ID NO: 1, or a Y100I W133A mutant of SEQ ID NO: 1. 
     
     
         15 . A fusion polypeptide comprising the mutant polypeptide of any one of  claims 1  to  14  operatively linked to a polypeptide of interest. 
     
     
         16 . The fusion polypeptide of  claim 15 , wherein the mutant polypeptide is further linked to the N terminus of the polypeptide of interest. 
     
     
         17 . The fusion polypeptide of any one of  claims 15  to  16 , wherein the mutant polypeptide is further linked to the C terminus of a further polypeptide of interest. 
     
     
         18 . The fusion polypeptide of any one of  claims 14  to  7 , wherein the polypeptide of interest and/or the further polypeptide of interest is a polypeptide selected from the group consisting of: a therapeutic polypeptide, a reporter polypeptide, an enzyme and a transcription factor. 
     
     
         19 . The fusion polypeptide of any one of  claims 14  to  18 , wherein the therapeutic polypeptide is a transcription factor, a neurotrophic factor, a cell surface receptor, an ATPase, a cyclin-dependent kinase inhibitor or an antibody. 
     
     
         20 . The fusion polypeptide of any one of  claims 14  to  19 , further comprising a signal peptide capable of causing secretion of the fusion polypeptide from a mammalian cell or a nuclear localisation signal capable of causing import of the fusion polypeptide in the nucleus. 
     
     
         21 . The fusion polypeptide of any one of  claims 14  to  20 , wherein the polypeptide of interest is additionally operatively linked to a reporter polypeptide. 
     
     
         22 . The fusion polypeptide of any one of  claims 14  to  21 , wherein the reporter polypeptide is a fluorescent protein, an enzyme or an affinity tag. 
     
     
         23 . A polynucleotide encoding the polypeptide of any one of  claims 1  to  14  or the fusion polypeptide of any one of  claims 15  to  22 . 
     
     
         24 . A gene expression system comprising a polynucleotide according to  claim 23 . 
     
     
         25 . A system for conditionally stabilising a fusion polypeptide comprising a polypeptide according to any one of  claims 15  to  22  and a ligand, wherein the ligand is capable of binding to the polypeptide and stabilising the fusion polypeptide. 
     
     
         26 . Use of the system according to  claim 25  to conditionally stabilise said polypeptide of interest. 
     
     
         27 . The use of  claim 26 , wherein the maximal activity of the polypeptide of interest in the presence of ligand is at least 9-fold higher than in the absence of ligand, such as at least 10-fold, such as at least 11-fold, such as at least 12-fold, such as at least 13-fold, such as at least 15-fold, such as at least 16-fold, such as at least 17-fold, such as at least 18-fold, such as at least 19-fold, such as at least 20-fold, such as at least 25-fold, such as at least 30-fold, such as at least 40-fold, such as at least 50-fold. 
     
     
         28 . The use of any one of  claims 26  to  27 , wherein the maximal activity of the polypeptide is determined by an assay such as an enzymatic assay, a spectrophotometric assay, a chemiluminescent assay, a calorimetric assay, a binding assay, a growth assay or a differentiation assay. 
     
     
         29 . The use of any one of  claims 26  to  28 , wherein the fusion polypeptide further comprises a reporter gene and wherein the activity of the polypeptide of interest is determined by measuring the activity of said reporter gene. 
     
     
         30 . The use of any one of  claims 26  to  29 , wherein the ligand is a dihydrofolate reductase inhibitor such as an antibiotic, preferably trimethoprim, brodimoprim, tetroxoprim, iclaprim, or pyrimethamine, or an anti-cancer drug, such as proguanil, methotrexate and pemetrexed, preferably trimethoprim. 
     
     
         31 . The use of  claim 30  wherein the dihydrofolate reductase inhibitor is an antibiotic such as trimethoprim, brodimoprim, tetroxoprim, iclaprim or pyrimethamine, preferably trimethoprim. 
     
     
         32 . A vector comprising a polynucleotide encoding the fusion polypeptide of any one of  claims 15  to  22 . 
     
     
         33 . The polypeptide of any one of  claims 1  to  14 , the fusion polypeptide of any one of  claims 15  to  22 , the system of  claim 25 , or the vector of  claim 32 , for use in the treatment of a disease in a subject in need thereof. 
     
     
         34 . The polypeptide, fusion polypeptide, system or vector for the use of  claim 33  wherein the disease is associated with reduced activity of the polypeptide of interest, or of a protein comprising the polypeptide of interest. 
     
     
         35 . The polypeptide, fusion polypeptide, system or vector for the use of any one of  claims 33  to  34 , wherein the treatment further comprises administering to the subject a ligand capable of binding to and stabilising the fusion polypeptide. 
     
     
         36 . An isolated host cell capable of expressing the fusion polypeptide of any one of  claims 15  to  22 . 
     
     
         37 . A method of treatment of a disease in a subject in need thereof, comprising administration of polypeptide of any one of  claims 1  to  14 , the fusion polypeptide of any one of  claims 15  to  22 , the system of  claim 25 , or the vector of  claim 32 .