C-terminal protein tagging

Abstract

In general, the invention features proteins having covalently bonded C-terminal puromycin tags and methods for their production.

What is claimed is:  
     
         1 . A protein having a covalently bonded puromycin tag, said tag being positioned at the C-terminal end of said protein.  
     
     
         2 . The protein of  claim 1 , wherein said tag is a small molecule.  
     
     
         3 . The protein of  claim 2 , wherein said small molecule is biotin.  
     
     
         4 . The protein of  claim 1 , wherein said tag is a detectable label.  
     
     
         5 . The protein of  claim 4 , wherein said detectable label is fluorescein, rhodamine, or BODIPY, or derivatives thereof.  
     
     
         6 . The protein of  claim 1 , wherein said tag is a functional group.  
     
     
         7 . The protein of  claim 1 , wherein said protein has a first functional group and said tag is a second functional group and wherein said first functional group has a reactivity orthogonal to the reactivity of said second functional group.  
     
     
         8 . The protein of  claim 1 , wherein said tag is a tether for attachment to a solid support.  
     
     
         9 . The protein of  claim 8 , wherein said solid support is a column, bead, or chip.  
     
     
         10 . The protein of  claim 1 , wherein said tag is one member of a specific binding pair.  
     
     
         11 . The protein of  claim 10 , wherein said tag is a phenyl diboronic acid derivative.  
     
     
         12 . The protein of  claim 1 , wherein said puromycin tag further comprises a nucleotide sequence positioned between said tag and said puromycin.  
     
     
         13 . The protein of  claim 12 , wherein said nucleotide sequence is between about 1-200 nucleotides in length.  
     
     
         14 . A method for C-terminal protein tagging, comprising 
 (a) providing a nucleic acid sequence encoding said protein;    (b) translating said sequence under conditions in which translation stalls at the 3′ end of said sequence, forming a stalled translation complex; and    (c) contacting said stalled translation complex with a puromycin tag under conditions in which said puromycin tag is covalently bonded to the C-terminus of said protein.    
     
     
         15 . The method of  claim 14 , wherein said tag is attached to the 5′-hydroxy group of said puromycin.  
     
     
         16 . The method of  claim 15 , wherein said tag is attached to the 5′-hydroxy group of said puromycin through a phosphate group.  
     
     
         17 . The method of  claim 14 , wherein said nucleic acid sequence encoding said protein contains no stop codons.  
     
     
         18 . The method of  claim 14 , wherein said translation step is carried out in the substantial absence of at least one translation release factor.  
     
     
         19 . The method of  claim 14 , wherein the 3′-end of said nucleic acid sequence encoding said protein is covalently linked to a DNA oligomer.  
     
     
         20 . The method of  claim 14 , wherein said tag is a small molecule.  
     
     
         21 . The method of  claim 20 , wherein said small molecule is biotin.  
     
     
         22 . The method of  claim 14 , wherein said tag is a detectable label.  
     
     
         23 . The method of  claim 22 , wherein said detectable label is fluorescein, rhodamine, or BODIPY, or a derivative thereof.  
     
     
         24 . The method of  claim 14 , wherein said tag is a functional group.  
     
     
         25 . The method of  claim 14 , wherein said protein has a first functional group and said tag is a second functional group and wherein said first functional group has a reactivity orthogonal to the reactivity of said second functional group.  
     
     
         26 . The method of  claim 14 , wherein said tag is a tether for attachment to a solid support.  
     
     
         27 . The method of  claim 26 , wherein said solid support is a column, bead, or chip.  
     
     
         28 . The method of  claim 14 , wherein said tag is one member of a specific binding pair.  
     
     
         29 . The method of  claim 28 , wherein said tag is a phenyl diboronic acid derivative.  
     
     
         30 . The method of  claim 14 , wherein said puromycin tag further comprises a nucleotide sequence positioned between said tag and said puromycin.  
     
     
         31 . The method of  claim 30 , wherein said nucleotide sequence is between about 1-200 nucleotides in length.