Method for producing circular or multimeric protein species in vivo or in vitro and related methods

Abstract

A method is disclosed for forming multimeric proteins. The method relies on intermolecular trans-splicing of a split intein either in vivo or in vitro.

1 . A method for producing a multimeric protein, comprising the steps of:
 (a) providing a first and a second target protein and a first and second intein fragment wherein the first intein fragment is fused at its C-terminus to the N-terminus of the first target protein and the second intein fragment is fused at its N-terminus to the C-terminus of the second target protein at its C-terminus; and   (b) permitting intermolecular trans-splicing of the first and second target proteins to produce the multimeric protein.   
     
     
         2 . A method according to  claim 1 , wherein the multimeric protein is produced in vitro. 
     
     
         3 . A method according to  claim 1 , wherein step (a) further comprises: introducing into a host cell, a first DNA encoding the first target protein and the first intein fragment and a second DNA encoding the second target protein and the second intein fragment. 
     
     
         4 . A method according to  claims 1  and  5 , wherein the target protein is selected from the group consisting of a peptide, a protein and an enzyme. 
     
     
         5 . A method according to  claim 1 , further comprising: attaching the first or the second target protein fused to an intein fragment, to an affinity resin via an affinity domain associated with the intein fragment. 
     
     
         6 . A method according to  claim 1 , wherein the affinity resin is formulated as an affinity-based support, a chip, a plate, a biochip support, a glass wafer or a microtiter plate. 
     
     
         7 . A method according to  claim 1 , wherein the intein fragments are derived from a naturally split intein. 
     
     
         8 . A method according to  claim 1 , wherein the intein fragments are derived from an artificially split intein. 
     
     
         9 . A method according to  claim 5 , wherein the affinity resin is selected from the group consisting of: a chitin-binding domain, the maltose-binding protein, a His tag, a cellulose-binding protein and a Flag-tag. 
     
     
         10 . A method according to  claim 1 , further comprising: adding a thiol reagent or altering pH or temperature for enhancing cleavage of the intein fragments from the target proteins. 
     
     
         11 . A method according to  claim 1 , further comprising: performing the trans-splicing reaction at an effective pH and temperature in the presence of a reducing agent to facilitate trans-splicing.