Small molecule-dependent inteins and uses thereof

Abstract

Elucidating the function of proteins in mammalian cells is particularly challenging due to the inherent complexity of these systems. Methods to study protein function in living cells ideally perturb the activity of only the protein of interest but otherwise maintain the natural state of the host cell or organism. Ligand-dependent inteins offer single-protein specificity and other desirable features as an approach to control protein function in cells post-translationally. Some aspects of this invention provide second-generation ligand-dependent inteins that splice to substantially higher yields and with faster kinetics in the presence of the cell-permeable small molecule 4-HT, especially at 37° C., while exhibiting comparable or improved low levels of back-ground splicing in the absence of 4-HT, as compared to the parental inteins. These improvements were observed in four protein contexts tested in mammalian cells at 37° C., as well as in yeast cells assayed at 30° C. or 37° C. The newly evolved inteins described herein are therefore promising tools as conditional modulators of protein structure and function in yeast and mammalian cells.

1 - 29 . (canceled) 
     
     
         30 . An estrogen-binding domain comprising the amino acid sequence NSLALSLTADQMVSALLDAEPPIL*YSEYD*PTSPFSEASMMGLLTNLADRELVHMINWAK RVPGFVDLTLHDQAHLLEC*AWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGM VEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRALDKITD TLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKYT*NVVPLYDLLLEM LDAHRLHA (SEQ ID NO: 13), wherein at least one of the residues L*, D*, C*, or T* is mutated. 
     
     
         31 . The estrogen-binding domain of  claim 30 , wherein the estrogen-binding domain comprises at least one of the following mutations: L*P, D*N, C*R, or T*K. 
     
     
         32 . The estrogen-binding domain of  claim 30 , wherein the estrogen-binding domain is fused to an N-terminal intein domain and a C-terminal intein domain, forming a ligand-dependent intein. 
     
     
         33 . The estrogen-binding domain of  claim 30 , wherein the N-terminal intein domain and the C-terminal intein domain are RecA-derived intein domains. 
     
     
         34 . The estrogen-binding domain of  claim 33 , wherein the N-terminal intein domain comprises the sequence CLAEGTRIFDPVTGTTHRIEDVVDGRKPIHVVAV*AKDGTLLARPVVSWFDQGTRDVIGLRI AGGAI*VWATPDHKVLTEYGWRAAGELRKGDRVA (SEQ ID NO: 10); and/or the N-terminal intein domain comprises the sequence RVQAFADALDDKFLHDMLAEE*LRYSVIREVLPTRRARTFDLEVEELHTLVAEGVVVHN (SEQ ID NO: 11). 
     
     
         35 . The estrogen-binding domain of  claim 34 , wherein at least one of amino acid residues V*, I*, or E* are mutated. 
     
     
         36 . The estrogen-binding domain of  claim 35 , wherein the intein comprises at least one of the following mutations: V*A, I*T, or E*G. 
     
     
         37 - 62 . (canceled)