Site-specific labeling of affinity tags in fusion proteins

Abstract

The present invention provides methods and fluorescent compounds that facilitate detecting and labeling of a fusion protein by being capable of selectively binding to an affinity tag. The fluorescent compounds have the general formula A(B)n, wherein A is a fluorophore, B is a binding domain that is a charged chemical moiety, a protein or fragment thereof and n is an integer from 1-6 with the proviso that the protein or fragment thereof not be an antibody or generated from an antibody. The present invention provides specific fluorescent compounds and methods used to detect and label fusion proteins that contain a poly-histidine affinity tag. These compounds have the general formula A(L)m(B)n wherein A is a fluorophore, L is a linker, B is an acetic acid binding domain, m is an integer from 1 to 4 and n is an integer from 1 to 6.

1 - 42 . (canceled) 
     
     
         43 . A fluorescent compound having formula A(L)m(B)n, wherein A is a fluorophore selected from the group consisting of borapolyazaindacene and coumarin, L is a linker, B is an acetic acid binding domain wherein said fluorescent compound contains at least three acetic acid groups that are capable of binding to a poly-histidine affinity tag, m is an integer from 1 to 4 and n is an integer from 1 to 6. 
     
     
         44 . The compound according to  claim 43 , wherein said linker is selected from the group consisting of —(CH 2 ) e C(X)NH(CH 2 ) e (NHC(X)(CH 2 ) e ) d —, —((C 6 R″ 4 )O) d (CH 2 ) e (C(X)NH(CH 2 ) e )(NH) d C(X)NH(C 6 R″ 4 )(CH 2 ) e — and —(O) d (CH 2 ) f O(C 6 R″ 4 )— wherein X is O or S, d is 0 or 1, e is 1 to 6, f is 2 or 3, and R″ is independently H, halogen, alkoxy or alkyl. 
     
     
         45 . The compound according to  claim 44 , wherein said acetic acid binding domain is selected from the group consisting of  − O 2 CCH(R)N(CH 2 CO −   2 ) 2 , —N(CH 2 CO 2 ) 2  and (CH 2 CO −   2 ) Z N[(CH(R)) S N(CH 2 CO −   2 )] T (CH(R)) S N(CH 2 CO −   2 ) Z  wherein Z is 1 or 2, S is 1 to 5, T is 0 to 4 and R is said linker. 
     
     
         46 . The compound according to  claim 45 , wherein said fluorophore is a borapolyazaindacene and said compound is selected from the group consisting of 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
       and salts thereof wherein R 30  may be the same or different and is selected from the group consisting of hydrogen, salt ion, —CH 2 OCOR 41  and an electron pair wherein R 41  is an alkyl group. 
     
     
         47 . The compound according to  claim 45 , wherein said fluorophore is a coumarin and said compound is selected from the group consisting of 
       
         
           
           
               
               
           
         
       
       and salts thereof wherein R 30  may be the same or different and is selected from the group consisting of hydrogen, salt ion, —CH 2 OCOR 41  and an electron pair wherein R 41  is an alkyl group. 
     
     
         48 . A composition comprising;
 a) a fluorescent compound capable of selectively binding, directly or indirectly, to affinity tag containing fusion protein, wherein said fluorescent compound comprises a fluorophore; and,   b) a fusion protein comprising an affinity tag, provided said fluorescent compound does not comprise an antibody or fragment thereof.   
     
     
         49 . The composition according to  claim 48 , wherein said fluorescent compound comprises a binding domain and a fluorophore selected from the group consisting of xanthene, cyanine, coumarin, acridine, anthracene, benzofuran, borapolyazaindacene and derivative thereof. 
     
     
         50 . The composition according to  claim 49  wherein said fluorescent compound is according to formula A(L)m(B)n wherein A is a fluorophore, B is an acetic acid binding domain wherein said compound comprises at least three acetic acid groups that are capable of selectively binding to a poly-histidine affinity tag, m is an integer from 1 to 4 and n is an integer from 1 to 6. 
     
     
         51 . The composition according to  claim 50 , wherein said composition further comprises a metal ion selected from the group consisting of nickel and cobalt. 
     
     
         52 . The composition according to  claim 49 , wherein said binding domain is glutathione.