J. Macak, R. Kupcik, P. Rehulka, P. Krulisova, Z. Bilkova
University of Pardubice,
Keywords: purification, proteins, His-tag, nanomaterial
Summary:Synthesis of biomolecules (e.g. proteins, polypeptides, oligosaccharides or nucleic acids) is of essential importance in life sciences as they are used for the development and production of efficient and selective biopharmaceuticals, including targeted bioactive therapeutics, recombinant proteins or vaccines. Extremely high purity of these biomolecules is required for in-vivo applications without risks of unexpected adverse side effects. Many recombinant proteins are synthesized with suitable affinity tags to facilitate the purification process. Immobilized-Metal Affinity Chromatography (IMAC) has been the most widely employed technique for separation of recombinant proteins. However, it has lot of drawbacks (mainly long processing time and low specificity). Within our presentation, we will introduce a completely new approach for purification of His-tagged recombinant proteins using novel inorganic composite and optimized separation conditions. The composite is based on self-organized anodic TiO2 nanotubes loaded homogenously with magnetite Fe3O4 nanoparticles. We will show that using this composite, His-tagged recombinant proteins can be extremely specifically isolated from the model as well as real mixture of proteins on behalf of other proteins, as demonstrated using electrophoresis and MALDI mass spectroscopy. Intriguing chelating properties of the composite, being used for the first time for such application, in combination with tailored binding and elution protocols, offer fascinating possibilities for specific isolation and elution of various recombinant polyhistidine-tagged proteins, not observed for any other carrier used for this purpose up to now. The achieved isolation protein´s specificity has a direct positive impact on the increased purification efficiency of proteins, lower costs of purification with very promising implications in life sciences. In particular, we will discuss details on the preparation of the composite, separation procedures and we will show purification results obtained using by this new approach.