Synthesis and performance of megaporous immobilized metal-ion affinity cryogels for recombinant protein capture and purification

Noor Shad Bibi, Naveen Kumar Singh, Roy N. Dsouza, Muhammad Aasim, Marcelo Fernández-Lahore
  • Journal of Chromatography A, January 2013, Elsevier
  • DOI: 10.1016/j.chroma.2012.11.036

Metal affinity chromatography for recombinant protein (his-tagged) capture and purification.

What is it about?

Megaporous cryogels with metal-ion affinity functionality possess enhanced protein-binding ability. These highly porous materials (pore sizes up to 100 μm) allowed the direct capture of a recombinant His6-tagged protein from a partially clarified extract. The total ligand density of the material was found to be 770 μmol/g. Application of a partially clarified cell extract in order to recover a His6-tagged protein (NAD(P)H-dependent 2-cyclohexen-1-one-reductase) yielded 12 mg of highly purified recombinant product per gram of adsorbent. Increased dynamic binding capacities were observed upon more significant degrees of grafting, although some reduction in the quality of the system hydrodynamics was also observed. Nevertheless, these immobilized metal-ion affinity cryogels show potential for a convenient single-step purification of recombinant proteins from raw cell extracts without the need for laborious pre-chromatographic sample clean-up procedures.

Why is it important?

Affinity chromatography is a widely used technique for the purification and polishing of bioproducts largely due to its high specificity. Megaporous IMAC cryogels provide a versatile, single-step method to recover proteins and other bioproducts from complex biological mixtures. Adsorbent materials of increased binding capacity are needed to ensure sufficient performance in such applications. Poly(methacrylic acid)-based cryogels can be tailored to allow for increased and highly specific binding capacities by exploiting affinity techniques such as IMAC. The varying degrees of grafting can affect binding capabilities and system hydrodynamics and have also provided a synthetic route to achieve different grafting densities. IMAC-functionalized cryogels offers enhanced protein-binding capacities. Pore sizes can be easily altered and offer pore sizes up to 100 μm. IMAC cryogels captures 12 mg/g of His-tagged protein.

Perspectives

Dr Naveen Kumar Singh
University of Notre Dame

This paper was well received and as a result, we were invited to publish a more detailed protocol in Methods in Molecular Biology.

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http://dx.doi.org/10.1016/j.chroma.2012.11.036

The following have contributed to this page: Professor Hector M. Fernandez Lahore and Dr Naveen Kumar Singh