Engineered E2 For Increasing The Content Of Free LYS11-Linked Ubiquitin

Abstract

The invention provides a chimeric E2 enzyme comprising a Ubc domain fused to a heterologous ubiquitin binding domain (UBD). The chimeric enzymes of the invention may be useful in producing elevated levels of free polyubiquitin.

What is claimed is: 
     
         1 . An E2 enzyme comprising a Ubc domain, from which an N-terminal tail or a C-terminal tail has been removed. 
     
     
         2 . An E2 enzyme according to  claim 1 , which is a chimeric enzyme wherein the Ubc is fused to a heterologous ubiquitin-binding domain (UBD). 
     
     
         3 . A chimeric E2 enzyme according to  claim 2 , wherein the UBD is C-terminal to the Ubc domain. 
     
     
         4 . A chimeric E2 enzyme according to  claim 2 , wherein the UBD is an α-helical, zinc finger or pleckstrin homology domain. 
     
     
         5 . A chimeric E2 enzyme according to  claim 2 , wherein the UBD is a domain selected from the group consisting of UIM, IUIM (MIU), DUIM, UBM, UBA, GAT, CUE, VHS, UBZ, NZF, ZnF A20, ZnF UBP (PAZ), PRU, GLUE, UEV, UBC, SH3, PFU and Jab1/MNP domains. 
     
     
         6 . A chimeric E2 enzyme according to  claim 4 , wherein the UBD is derived from Isopeptidase T. 
     
     
         7 . A chimeric E2 enzyme according to  claim 6 , wherein the UBD comprises the sequence from about position 163 to about position 291 of Isopeptidase T. 
     
     
         8 . A chimeric E2 enzyme according to  claim 4 , wherein the UBD is a UBA, UIM, ZnF or NZF domain. 
     
     
         9 . An E2 enzyme according to  claim 1 , wherein the Ubc domain is derived from an E2 enzyme selected from the group consisting of UBE2A, UBE2B, UBE2C, UBE2D1, UBE2D2, UBE2D3, UBE2D4, UBE2E1, UBE2E2, UBE2E3, UBE2F, UBE2G1, UBE2G2, UBE2H, UBE2I, UBE2J1, UBE2J2, UBE2K, UBE2L3, UBE2L6, UBE2M, UBE2N, UBE2NL, UBE2O, UBE2Q1, UBE2Q2, UBE2R1, UBE2R2, UBE2S, UBE2T, UBE2U, UBE2W, UBE2Z and BIRC6. 
     
     
         10 . An E2 enzyme according to  claim 9 , wherein the E2 enzyme is a class II E2 enzyme. 
     
     
         11 . An E2 enzyme according to  claim 10 , wherein an N-terminal or a C-terminal amino acid tail on the class II E2 enzyme is replaced by the UBD. 
     
     
         12 . An E2 enzyme according to  claim 10  or  claim 11 , wherein the Ubc domain is derived from UBE2S. 
     
     
         13 . An E2 enzyme according to  claim 12 , wherein the Ubc domain comprises residues 1 to 156 of UBE2S. 
     
     
         14 . A method for increasing the capacity of an E2 enzyme to produce free polyubiquitin chains in solution, comprising conjugating fusing the Ubc domain of said E2 enzyme to a UBD. 
     
     
         15 . A method according to  claim 14 , wherein the E2 enzyme is selected from the group consisting of UBE2A, UBE2B, UBE2C, UBE2D1, UBE2D2, UBE2D3, UBE2D4, UBE2E1, UBE2E2, UBE2E3, UBE2F, UBE2G1, UBE2G2, UBE2H, UBE21, UBE2J1, UBE2J2, UBE2K, UBE2L3, UBE2L6, UBE2M, UBE2N, UBE2NL, UBE20, UBE2Q1, UBE2Q2, UBE2R1, UBE2R2, UBE2S, UBE2T, UBE2U, UBE2V1, UBE2V2, UBE2V3, UBE2W, UBE2Z, AKTIP and BIRC6 and the UBD is a domain selected from the group consisting of UIM, IUIM (MIU), DUIM, UBM, UBA, GAT, CUE, VHS, UBZ, NZF, A20-like ZnF, ZnF UBP (PAZ), PRU, GLUE, UEV, UBC, SH3, PFU and Jab1/MNP domains. 
     
     
         16 . A method according to  claim 15 , wherein the E2 enzyme is UBE2S. 
     
     
         17 . A method according to  claim 14 , wherein the UBD is a ZnF UBP domain. 
     
     
         18 . A method for producing free polyubiquitin chains linked through a desired lysine residue, comprising the steps of: (a) selecting an E2 enzyme which possesses the desired lysine residue specificity; (b) fusing the Ubc catalytic domain of said E2 enzyme to a UBD ubiquitin binding domain; and incubating the resulting chimeric protein with an E1 ubiquitin activating enzyme and monomeric ubiquitin. 
     
     
         19 . A method according to  claim 18 , wherein the incidence of undesired lysine linkages is reduced by including a linkage-specific deubiquitinase in the incubation.