US2015344549A1PendingUtilityA1

Split inteins, conjugates and uses thereof

61
Assignee: UNIV PRINCETONPriority: Jun 27, 2012Filed: Jun 24, 2013Published: Dec 3, 2015
Est. expiryJun 27, 2032(~5.9 yrs left)· nominal 20-yr term from priority
Inventors:Tom W. MuirMiquel Vila-PerelloZhihua LiuNeel H. Shah
C12P 21/02C12N 15/62C12N 9/93C07K 2319/92C07K 2319/50C07K 14/195C12P 21/06C07K 16/18
61
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Claims

Abstract

Disclosed herein are split inteins, fused proteins of split inteins, and methods of using split inteins to efficiently purify and modify proteins of interest. Thus, provided herein are fusion proteins of a polypeptide and a split intein N-fragment, or variant thereof, as described below in greater detail. Also provided are complexes of the fusion protein and a split intein C-fragment or variant thereof as described in detail below. The complex of the fusion protein and C-fragment or variant thereof can be via a covalent interaction between the fusion protein and C-fragment or variant or via a noncovalent interaction (e.g., ionic, H-bonding, and/or van der Waals interaction). Further provided herein are split intein C-fragments or variants thereof.

Claims

exact text as granted — not AI-modified
1 . A fusion protein comprising a split intein N-fragment and a polypeptide, wherein the split intein N-fragment comprises a sequence selected from the group consisting of SEQ ID NOs: 1-19, or a variant thereof. 
     
     
         2 . The fusion protein of  claim 1 , wherein the split intein N-fragment comprises a sequence of SEQ ID NO: 19, or a variant thereof. 
     
     
         3 . The fusion protein of  claim 1 , wherein the split intein N-fragment comprises a sequence selected from the group consisting of SEQ ID NO: 20-38 and 761, or a variant thereof. 
     
     
         4 . The fusion protein of  claim 1 , wherein the polypeptide has a molecular weight of 40 kDa or greater. 
     
     
         5 . The fusion protein of  claim 4 , wherein the polypeptide is an antibody or fragment thereof. 
     
     
         6 . The fusion protein of  claim 5  wherein the antibody is an IgG antibody or fragment thereof. 
     
     
         7 . The fusion protein of  claim 6 , wherein a heavy chain of the antibody is fused to the split intein N-fragment. 
     
     
         8 . The fusion protein of  claim 7 , wherein each heavy chain of the antibody is fused to the split intein N-fragment. 
     
     
         9 . The fusion protein of  claim 6 , wherein a light chain of the antibody is fused to the split intein N-fragment. 
     
     
         10 . The fusion protein of  claim 1 , wherein the polypeptide is secreted from a cell. 
     
     
         11 . A complex comprising the fusion protein of  claim 1  and the split intein C-fragment of  claim 12 . 
     
     
         12 . A split intein C-fragment comprising a sequence selected from the group consisting of SEQ ID NOs: 57-74, or a variant thereof. 
     
     
         13 . The split intein C-fragment of  claim 12  comprising a sequence selected from the group consisting of SEQ ID NOs: 75-128, or a variant thereof. 
     
     
         14 . The split intein C-fragment of  claim 12  comprising a sequence of SEQ ID NO: 707, or a variant thereof. 
     
     
         15 . The split intein C-fragment of  claim 14  comprising a sequence selected from the group consisting of SEQ ID NOs: 708-711, or a variant thereof. 
     
     
         16 . The split intein C-fragment of  claim 12  attached to a support. 
     
     
         17 . The split intein C-fragment of  claim 16 , wherein the support comprises a particle, a bead, a resin, or a slide. 
     
     
         18 . The split intein C-fragment of  claim 17 , wherein the split intein C-fragment comprises a sequence selected from the group consisting of SEQ ID NOs: 417-704 and 745-759, or a variant thereof. 
     
     
         19 . A method comprising
 (a) contacting (1) a fusion protein comprising a polypeptide and a split intein N-fragment comprising a sequence selected from the group consisting of SEQ ID NO: 1-19, or a variant thereof, and (2) a split intein C-fragment comprising a sequence selected from the group consisting of SEQ ID NO: 57-74, or a variant thereof, wherein contacting is performed under conditions that permit binding of the split intein N-fragment to the split intein C-fragment to form an intein intermediate; and   (b) contacting the intein intermediate with a nucleophile to form a conjugate of the protein and the nucleophile.   
     
     
         20 - 34 . (canceled)

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