A novel fusion tag for recombinant proteins purification

A. M. TARGOVNIK; M. C. MARTÍNEZ-CERON; N. URTASUN; O. CASCONE; M. V. MIRANDA; S. A. CAMPERI

Tucumán, Argentina

Congreso; XLV Reunión Anual SAIB; 2009

SAIB: Sociedad Argentina de Investigación Bioquímica y Biología Molecular

Affinity chromatography (AC) is the most effective method for direct isolation and purification of biomolecules from complex mixtures. Short peptides are excellent ligands for AC as they are not likely to cause an immune response in case of leakage into the product, they are more stable than antibodies to elution and cleaning conditions and they usually have very acceptable selectivity. Hydropathically complementary peptides show enough selectivity to be successfully used as ligands for protein purification in AC. In this work we design a system for recombinant protein purification using as model the Green Fluorescent Protein (GFP) expressed in E. coli and two complementary peptides: The peptide LSAAIIIPFLLH was synthesised by solid phase using the Fmoc chemistry and immobilised on NHS-agarose and GFP was expressed with the peptide KNYPKKKMEKRF as a fusion tag in the carboxi termini. After cell disruption, the extract was directly applied to the complementary peptide-agarose chromatographic matrix equilibrated with 20 mM sodium phosphate buffer, pH 7.0. The GFP fused with the peptide tag was adsorbed and eluted specifically with 50 mM sodium acetate buffer, pH 3.0, 1 M Tris. The method designed can be applied for purification of other recombinant proteins.