Research Projects
2. Application of Immobilised Metal Affinity Chromatography for the purification of proteins
(collaboration with Prof. Milton Hearn, Centre for Green Chemistry)
Stringent requirements on the purity of pharmaceutically important proteins such as interferon, vaccines and antibodies as well as the growing interest in medical and biotechnological research in separating bio-molecules impose a need to handle the purification of these proteins from complex mixtures in highly efficient and predictable manners.
Amongst many separation techniques Immobilised Metal Affinity Chromatography (IMAC) has proven to be an efficient and versatile technology for the isolation and purification of industrial enzymes and of proteins that are of commercial importance or used in research fields such as genetics, molecular biology and biochemistry. IMAC uses immobilised metal complexes, formed by reacting metal ions with chelating ligands that are attached to a chromatographic support (e.g. Sepharose). The capture of proteins is possible through interaction of specific metal binding sites (amino acid side chains on protein surface or specific developed tags, attached to the protein). The selectivity of IMAC can be influenced by the choice of metal ion, chelating ligand, absorption and elution conditions, or by modification of the target protein.
A schematic diagram showing protein adsorption and desorption from the matrix is shown below.
L = ligand M = metal
Traditionally, ligands like iminodiacetic acid (IDA) and nitrilotriacetic acid (NTA) complexed with Cu(II) or Ni(II) have been used widely and gels incorporating these ligands are available commercial suppliers (Amersham Pharmacia). Interested in extending the versatility of IMAC, we have been applying macrocyclic ligands (see diagram below) with a view to increase the stability of the immobilised complexes thereby minimising metal leakage during purification and expanding the range of biomolecules that can be purified by IMAC. Many exciting applications of these complexes can be envisaged, the purification of proteins prepared by recombinant techniques being one example. Towards this end, we have also attached affinity tags to the C- or N-terminus of various proteins containing metal binding groups (e.g. His for Cu2+) to allow single step purification (Jane Mooney, PhD student). In a project funded by the Australian Research Council and Novo Nordisk Dr. Phei Lok, Dr Rachel Daly and Ute Kreher (PhD Student) have been exploring ligands that will strongly bind to hard metal ions (Ca2+, Fe3+, etc.) enabling them to be used in IMAC.
Recent publications
[1] Peptide purification by means of metal ion affinity chromatography. T. Christensen, M.T.W. Hearn, L. Spiccia, W. Jiang, J.N. Mooney, B. Graham, PCT International. Application 2003, WO 03042249. |
