INVESTIGADORES
CAMPERI Silvia Andrea
artículos
Título:
Fully automated screening of a combinatorial library to avoid false positives: application to tetanus toxoid ligand identification
Autor/es:
M. C. MARTÍNEZ-CERON; L ÁVILA; S.L. GIUDICESSI; J. M. MINOIA; M. FINGERMANN; S.A. CAMPERI; F. ALBERICIO; O. CASCONE
Revista:
ACS omega.
Editorial:
ACS
Referencias:
Lugar: Washington; Año: 2021
ISSN:
2470-1343
Resumen:
Peptide ligands are widely used in protein purification by affinity chromatography. Here we applied a fully automated two-stage library screening method, that avoids false positive peptidyl-bead selection and applied it to tetanus toxoid purification. The first library screening was performed using only rhodamine (a fluorescent dye) and fluorescent beads were isolated automatically flow cytometry and discarded. A second screening was then performed with the rest of the library, using the target protein (tetanus toxoid)-rhodamine conjugate. This time, fluorescent beads were selected and peptide sequences identified by MALDI MS/MS. Those appearing with greater frequency were synthesized and immobilized on agarose to evaluate a range of chromatographic purification conditions. The affinity matrix PTx1-agarose (Ac-Leu-Arg-Val-Tyr-His-Gly-Gly-Ala-Gly-Lys-agarose) showed the best performance when 20 mM Sodium Phosphate, Tween 20 0.05%, pH 5.9 as adsorption buffer and 100 mM Tris-HCl, 100 mM NaCl, pH 8.0 as elution buffer were used. Pure tetanus toxoid (Ttx) was loaded on a chromatographic column filled with PTx1 matrix, and 96% adsorption was achieved with a Kd of 9.18 ± 0.07 nmol/L and a qm of 1.31 ± 0.029μmol Ttx/mL matrix. Next, Clostridium tetani culture supernatant treated with formaldehyde (to obtain the toxoid) was applied as a sample. The SDS-PAGE analysis showed a band, identified by ESI MS as Ttx, that appeared only in the elution fraction, where S-layer protein was also detected.