INVESTIGADORES
CAMPERI Silvia Andrea
congresos y reuniones científicas
Título:
Recombinant Protein Purification Using Complementary Peptides as Affinity Tags.
Autor/es:
M. C. MART├ŹNEZ-CERON; A. M. TARGOVNIK; N. URTASUN; M. V. MIRANDA; O. CASCONE; S. A. CAMPERI
Reunión:
Simposio; 14th International Biotechnology Symposium and Exhibition.; 2010
Resumen:
Affinity tags have become highly popular tools for purifying recombinant proteins from crude extracts. Besides, short peptides are excellent ligands for affinity chromatography, 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 designed de novo show enough selectivity to be used successfully as peptide ligands for protein purification from crude extracts. Recognition specificity and selectivity in the interaction between the complementary peptide pair LSAAIIIPFLLH and KNYPKKKMEKRF have been demonstrated by other authors. In this work we designed a recombinant protein purification method using a peptide affinity tag that binds to a peptide-binding partner immobilised on a chromatographic matrix using the green fluorescent protein (GFP) expressed in E. coli as the model. The peptide LSAAIIIPFLLH was synthesised by solid phase using the Fmoc chemistry and immobilised in NHS-agarose. The GFP was expressed either with a fusion tag KNYPKKKMEKRF on the carboxy terminus or without a fusion tag. After cell disruption, the extract was directly applied to a LSAAIIIPFLLH-agarose column using 20 mM sodium phosphate buffer, pH 7.0, for the adsorption step. The GFP fused with the peptide tag KNYPKKKMEKRF was adsorbed and then eluted specifically with 1 M Tris . The yield was 98% and the purification factor 4.6. On the other hand, GFP without tag pass through without interacting with the peptide-agarose column. The method designed can be applied for the purification of other recombinant proteins.
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