Method for selective derivatization of oligohistidine sequence of recombinant proteins

Patent metadata

Field	Value
Publication number	US-9441010-B2
Application number	US-201313962426-A
Country	US
Kind code	B2
Filing date	Aug 8, 2013
Priority date	Aug 8, 2012
Publication date	Sep 13, 2016
Grant date	Sep 13, 2016

Abstract

Methods and compositions for the selective derivatization of a oligohistidine-tagged recombinant protein. A modifying compound comprised of an imidazole reactive group, a linker, and a ligating group is contacted with the recombinant protein, and a cooperative bond forms between the ligating group and the oligohistidine tag in the presence of a metal cation, and a covalent bond forms between the imidazole reactive group and an imidazole ring of the oligohistidine tag followed by the concomitant separation of the imidazole reactive group from the linker. Addition of a metal chelator results in the dissociation of the ligating group and the oligohistidine tag.

First claim

What is claimed is:

1. A method for the selective derivatization of a recombinant protein, the method comprising:
contacting a recombinant protein comprising an oligohistidine tag with a modifying compound in the presence of a metal cation, wherein the modifying compound comprises the formula X 1 X 2 X 3 , wherein:
X 1 is an imidazole reactive group comprising the structure represented by the formula:

wherein R1 is an electron withdrawing group and R2 is a connector connecting the imidazole reactive group to X 2 ;

X 2 is a linking group; and
X 3 is at least one ligating group;
wherein said contacting is performed under conditions sufficient for the formation of a bond between X 3 and the oligohistidine tag utilizing said metal cation, and under conditions sufficient for the formation of a covalent bond between X 1 and an imidazole ring of the oligohistidine tag and concomitant separation of X 1 and X 2 ; and
breaking the bond between X 3 and the oligohistidine tag using a metal chelator.

2. The method of claim 1 , wherein said electron withdrawing group is selected from the group consisting of an acyl group, an ester group, an alkylsulfonyl group, an arylsulfonyl group, a nitro group, a cyano group, a carbamoyl group, and combinations thereof.

3. The method of claim 1 , wherein X 3 comprises a plurality of ligating groups.

4. The method of claim 1 , wherein X 3 is selected from the group consisting of a primary amino group, a secondary amino group, a teriary amino group, a carboxy group, an imino group, a phenol group, a catechol group, a hydroxamate group, a sulfamido group, a phosphoryl group, an imidazolyl group, a thiazolyl group, an oxazolyl group, a pyridazolyl group, a pyridyl group, a thiol group, and combinations thereof.

5. The method of claim 1 , wherein said metal cation is selected from the group consisting of Ni 2+ , Zn 2+ , Cu 2+ , Fe 2+ , Co 2+ , Co 3+ , Fe 3+ , and combinations thereof.

6. The method of claim 1 , wherein said conditions sufficient for the formation of a bond between X 3 and the oligohistidine tag comprise the presence of an additive.

7. The method of claim 6 , wherein the additive comprises imidazole, N-methylimidazole, or N-alkylimidazole.

8. A compound for the selective derivatization of a recombinant protein comprising an oligohistidine tag, the compound comprising:
the formula X 1 X 2 X 3 , wherein X 1 is an imidazole reactive group comprising the structure represented by the formula:

wherein R1 is an electron withdrawing group and R2 is a connector connecting the imidazole reactive group to X 2 ;

X 2 is a linking group; and
X 3 is at least one ligating group;
wherein said compound is adapted to form a bond between X 3 and a oligohistidine tag of a recombinant protein in the presence of a metal cation, and further adapted to form a covalent bond between X 1 and an imidazole ring of the oligohistidine tag.

9. The compound of claim 8 , wherein X 3 comprises a plurality of ligating groups.

10. The compound of claim 8 , wherein X 3 is selected from the group consisting of a primary amino group, a secondary amino group, a teriary amino group, a carboxy group, a phosphoryl group, an imidazolyl group, a thiazolyl group, an oxazolyl group, a pyridazolyl group, a pyridyl group, and combinations thereof.

11. A method for the selective derivatization of a recombinant protein, the method comprising:
contacting a recombinant protein comprising an oligohistidine tag with a modifying compound in the presence of a metal cation, wherein the modifying compound comprises the formula
X 1 X 2 X 3 ,
wherein
X 1 is an imidazole reactive group comprising the structure represented by the formula below:

wherein R1 is an electron withdrawing group selected from the group consisting of an acyl group, an ester group, an alkylsulfonyl group, an arylsulfonyl group, a nitro group, a cyano group, and combinations thereof, and R2 is a linker to a linking group;

X 2 is said linking group; and
X 3 is a plurality of ligating groups each selected from the group consisting of a primary amino group, a secondary amino group, a teriary amino group, a carboxy group, a phosphoryl group, an imidazolyl group, a thiazolyl group, an oxazolyl group, a pyridazolyl group, a pyridyl group, and combinations thereof;
and further wherein said contacting is performed under conditions sufficient for the formation of a bond between X 3 and the oligohistidine tag utilizing a metal cation, and under conditions sufficient for the formation of a covalent bond between X 1 and an imidazole ring of the oligohistidine tag and concomitant separation of X 1 and X 2 ; and
breaking the bond between X 3 and the oligohistidine tag using a metal chelator.

Assignees

• Melman Artem
• Univ Clarkson

Inventors

• Melman Artem

Classifications

• C07K1/107 Primary

  by chemical modification of precursor peptides · CPC title

• C07K1/13 Primary

  Labelling of peptides · CPC title

• C07C69/736

  the hydroxy group of the ester being etherified with a hydroxy compound having the hydroxy group bound to a carbon atom of a six-membered aromatic ring · CPC title