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Response (RU)201816141210864200 5e-6 1e-5 1,5e-5 2e-5 2,5e-5 3e-5Concentration MFig. 1. A representative affinity analysiswith Biacore T100 of rhEPO with apositive peptide.with either Texas Red or biotin, as describedpreviously by Marani et al. [6,7].Fluorescent beads or beads showing apositive reaction with streptavidinperoxidasewere isolated. Fifty beadsshowed a positive reaction. Peptides werecleaved from each bead with NH 4 OH, elutedwith AcOH/MeCN/H 2 O (3/4/3) andsequenced by tandem MALDI-TOF-MS.Those sequences showing greater consensuswere synthesized and their affinity to rhEpowas evaluated using a plasma resonancebiosensor (Biacore T100). Kd valuesbetween 10 -5 -10 -6 M were obtained(Figure 1). Peptides with the highest affinitywere immobilized on NHS-agarose. All peptide-agarose matrices showed affinity for rhEpo(Figure 2A). Also, the affinity of the peptide-agarose matrices for bovine seroalbumin(BSA) - usually present in the culture supernatants - was assessed (Figure 2B). Thosepeptides with the highest selectivity between rhEpo and BSA were chosen for futuredevelopment of a chromatographic matrix for rhEPO purification.Absorbance at 280 nm0.350.3 A0.250.20.150.10.0500 20 40 60Volume (mL)Absorbance at 280 nm00 10 20 30Volume (mL)Fig. 2. Agarose-positive peptide column chromatogram of A) rhEpo and B) SAB. Afterapplying the protein solution, the column was washed with the equilibrating buffer (20mM sodium phosphate, pH 4.0). Elution was performed with 100 mM sodium acetatebuffer, pH 3.0, 0.5 M NaCl. The rhEpo was adsorbed while the SAB pass through withoutinteraction with the column. The arrow indicates the buffer change.AcknowledgementsThis study was partially supported by the Consejo Nacional de Investigaciones Científicas y Técnicas(CONICET: PIP 00230, PIP 00052), the University of Buenos Aires (UBA: B 043), the AgenciaNacional de Promoción Científica y Tecnológica de la República Argentina (ANPCyT: PICT 32309),CICYT (CTQ2006-03794/BQU, CTQ2008-00177), and the Generalitat de Catalunya (2005SGR00662). M.M.M, M. E., O.C. and S.A.C. are researchers of the CONICET. We thank Simon Côté fromMatrix Innovation Inc. for kindly donating HMBA-ChemMatrix.References1. Jelkmann, W. Br. J. Haematol. 141, 287-297 (2008).2. Roque, A.C., Lowe, C.R. Meth. Mol. Biol. 421, 1-21 (2008).3. Tozzi, C., Anfossi, L., Giraudi, G. J. Chromatogr. B 797, 289-304 (2003).4. Lam, K.S., Salmon, S.E., Hersh, E.M., Hruby, V.J., Kazmierski, W.M. Nature 354, 82-84 (1991).5. Furka, A., Sebestyen, F., Asgedom, M., Dibo, G. Int. J. Peptide Protein Res. 37, 487-493 (1991).6. Marani, M.M., de Oliveira, E., Côte, S., Camperi, S.A., Albericio, F., Cascone, O. Anal. Biochem.370, 215-222 (2007).7. Marani, M.M., Martínez Ceron, M.C., Giudicessi, S.L., de Oliveira, E., Côté, S., Erra-Balsells, R.,Albericio, F., Cascone, O., Camperi, S.A. J. Comb. Chem. 11, 146-150 (2009).8. Martínez-Ceron, M.C., Giudicessi, S.L., Marani, M.M., Albericio, F., Cascone, O., Erra-Balsells,R., Camperi, S.A. Anal. Biochem. 400, 295-297 (2010).1.210.80.60.40.2B195