Purification of Histidine-Tagged Proteins Using Nitrilotriacetic Acid Microgels
Inventors: Daniel Kohane, Boaz Mizrahi
Invention Types: Research Tool
Research Areas: Metabolic Disease, Stem Cell
Keywords: Assay, Drug Discovery, ReagentFor More Information Contact: Khunkhun, Rajinder
Immobilized metal affinity chromatography (IMAC) is the most frequently used method for the separation and purification of histidine-tagged (his-tagged) proteins. In this technique, the high affinity of metal ions such as nickel or cobalt to a tag sequence on the protein of interest creates strong yet reversible binding. A major limitation of current systems is their inefficiency in purifying many recombinant proteins, particularly when present in their native state or in low concentrations in the cell lysate. Performance deficiencies are caused in part by the clogging or destruction of matrix micropores by undissolved salts and other compounds during particle synthesis, limiting the surface area accessible for binding. Low surface metal density can further impair efficiency. |
The current invention is a straightforward synthetic scheme to produce microgel particles where the metal-chelating moiety, nitrilotriacetic acid (NTA), is distributed throughout the entire matrix. This new type of microgel particle is characterized by higher nickel density (inside and on the surface) than conventional beads leading to a very large area of exposure. The gels aim to overcome the deficiencies of current methods by eliminating a separate coating step, hence reducing the clogging of matrix pores during synthesis, and by further improving the efficiency of his-tagged protein purification by enhancing the penetration of proteins into the matrix while presenting a high density of metal ions.
This approach may be applicable to a wide variety of technologies involving immobilization and separation of biological molecules, as well as surface coating.
In this invention, protein-binding microgels were successfully synthesized from NTAs and other monomers. They were produced by two simple synthetic steps amenable to large-scale production, which may reduce the high costs associated with many protein purification systems. Comparing these microgels with commercially available HisTaq beads showed that the same amount of microgel could bind more than 3.5 times the amount of protein.||
The versatile acrylic backbone will allow easy tuning of particle properties to modify performance as desired. When loaded with Ni+2, the microgels offer an efficient alternative to current methods of enriching, immobilizing and purifying low and high molecular weight proteins and possibly other biomolecules.
IPStatus: Pat. Pend.