Please use this identifier to cite or link to this item: http://sgc.anlis.gob.ar/handle/123456789/2354
Title: Fully Automated Screening of a Combinatorial Library to Avoid False Positives: Application to Tetanus Toxoid Ligand Identification
Authors: Martínez Ceron, María C 
Ávila, Lucía 
Giudicessi, Silvana L 
Minoia, Juan M 
Fingermann, Matías 
Camperi, Silvia A 
Albericio, Fernando 
Cascone, Osvaldo 
Issue Date: 27-Jul-2021
Journal: ACS omega 
Abstract: 
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 sulforhodamine (a fluorescent dye), and fluorescent beads were isolated automatically by 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 isolated, and peptide sequences were identified by matrix-assisted laser desorption/ionization tandem mass spectrometry. 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, 0.05% Tween 20, pH 5.9 as adsorption buffer and 100 mM Tris-HCl, 100 mM NaCl, pH 8.0 as elution buffer were used. A pure tetanus toxoid (Ttx) was loaded on a chromatographic column filled with the PTx1 matrix, and 96% adsorption was achieved, with a K d of 9.18 ± 0.07 nmol/L and a q m of 1.31 ± 0.029 μmol Ttx/mL matrix. Next, a Clostridium tetani culture supernatant treated with formaldehyde (to obtain the toxoid) was applied as a sample. The sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed a band, identified by electrospray ionization mass spectrometry as the Ttx, that appeared only in the elution fraction, where an S-layer protein was also detected.
Description: 
Fil: Martínez Cerón, María C. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica, Cátedra de Biotecnología. Instituto de Nanobiotecnología (NANOBIOTEC). Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). Buenos Aires; Argentina.

Fil: Ávila, Lucía. ANLIS "Dr. Carlos G. Malbrán". Instituto Nacional de Producción de Biológicos. Buenos Aires; Argentina.

Fil: Giudicessi, Silvana L. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica, Cátedra de Biotecnología. Instituto de Nanobiotecnología (NANOBIOTEC). Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). Buenos Aires; Argentina.

Fil: Minoia, Juan M. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica, Cátedra de Biotecnología. Instituto de Nanobiotecnología (NANOBIOTEC). Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). Buenos Aires; Argentina.

Fil: Fingermann, Matías. ANLIS "Dr. Carlos G. Malbrán". Instituto Nacional de Producción de Biológicos. CONICET. Buenos Aires; Argentina.

Fil: Camperi, Silvia A. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica, Cátedra de Biotecnología. Instituto de Nanobiotecnología (NANOBIOTEC). Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). Buenos Aires; Argentina.

Fil: Albericio, Fernando. Universidad de Barcelona. Centro de Networking en Bioingeniería, Biomateriales y Nanomedicina. Departamento de Química Orgánica. Instituto de Química Avanzada de Cataluña. Barcelona; España.
Universidad de Kwazulu-Natal. Facultad de Química y Física. Durban; Sudáfrica.

Fil: Cascone, Osvaldo. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica, Cátedra de Biotecnología. Instituto de Nanobiotecnología (NANOBIOTEC). Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). ANLIS "Dr. Carlos G. Malbrán". Instituto Nacional de Producción de Biológicos. Buenos Aires; Argentina.
URI: http://sgc.anlis.gob.ar/handle/123456789/2354
DOI: 10.1021/acsomega.1c01814
Appears in Collections:Publicaciones INPB

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