Recombinant protein purification using complementary peptides as affinity tags

MARÍA CAMILA MARTINEZ CERON; ALEXANDRA M. TARGOVNIK; NICOLAS URTASUN; OSVALDO CASCONE; MARÍA VICTORIA MIRANDA; SILVIA CAMPERI

NEW BIOTECHNOLOGY

ELSEVIER SCIENCE BV

Lugar: Amsterdam; Año: 2012 vol. 29 p. 206 - 210

1871-6784

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 His-Leu-Leu-Phe-Pro-Ile-Ile-Ile-Ala-Ala-Ser-Leu and Lys-Asn-Tyr-Pro-Lys-Lys-Lys-Met-Glu-Lys-Arg-Phe 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 immobilized on a chromatographic matrix using the enhanced green fluorescent protein expressed (EGFP) in E. coli as the model. The peptide Gly-Gly-Gly-His-Leu-Leu-Phe-Pro-Ile-Ile-Ile-Ala-Ala-Ser-Leu was synthesised by solid phase using the Fmoc chemistry and immobilised in NHS-Sepharose (PC-Sepharose). Gly residues were added as a spacer arm at the N terminus. The EGFP was expressed either with the fusion tag Lys-Asn-Tyr-Pro-Lys-Lys-Lys-Met-Glu-Lys-Arg-Phe on the C terminus (EGFP-CPTag) or without a fusion tag. After cell disruption, the extract was directly applied to the PC-Sepharose column using 20 mM sodium phosphate buffer, pH 7.0, for the adsorption step. The EGFP-CPTag was adsorbed and then eluted specifically with 1 M Tris. The yield was 98% and the purification factor 4.6. On the other hand, EGFP without tag pass through without interacting with the PC-Sepharose column. The method designed can be applied for the purification of other recombinant proteins.