A novel hGM CSF derived tag for immuno-detection of recombinant proteins

PEROTTI, N.; KRATJE, R. B.; ETCHEVERRIGARAY, M.; OGGERO EBERHARDT, M.

Pinamar. Pcia. Buenos Aires. Argentina

Congreso; X Congreso de la Panamerican Association of Biochemistry and Molecular Biology (PABMB) ? XLI Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB) ? XX Reunión Anual de la Sociedad Argentina de Neuroquímica.; 2005

Panamerican Association of Biochemistry and Molecular Biology; Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; Sociedad Argentina de Neuroquímica

Epitope tagging with peptides has become an important tool for detecting, localizing and purifying recombinant proteins. Moreover, the development of small tags acquires high relevance for fusion protein design where its functionality should not be affected by tag addition. A continuous epitope A1PAR4, derived from human Granulocyte-Macrophage Colony-Stimulating Factor (hGM-CSF), was delineated by an overlapping peptide scan employing an anti-cytokine monoclonal antibody (mAb CC1H7). The peptide APAR was fused to the N and C- terminal region of the recombinant human interferon alfa2b (rhIFN alfa2b) using several linkers (APARGGG, GGGAPAR and APARSPS, being SPS the natural consecutive amino acid sequence of APAR in the GM CSF molecule). Fusion proteins were expressed in eukaryotic (CHO.K1 cells) and prokaryotic (E. coli) systems. Only APARGGG and APARSPS carrying proteins were recognized by mAb CC1H7. Both proteins were strongly recognized by western blot but only APARSPS fused protein was specifically recognized by methods that evaluate native proteins. These features suggest the use of the epitope APARSPS as a tag for fusion protein design, conferring the property of being recognized by procedures that preserve the native conformation as well as by others that promote the partial or complete denaturalization.