Peptide affinity chromatography based on combinatorial strategies for protein purification

SILVIA A. CAMPERI; M. CAMILA MARTÍNEZ CERON; SILVANA L. GIUDICESSI; MARIELA M. MARANI; FERNANDO ALBERICIO; OSVALDO CASCONE

Methods in Molecular Biology book: Protein Downstream Processing

Humana Press

Año: 2014; p. 277 - 302

We describe a method to develop affinity chromatography matrices with short peptide ligands for protein purification. The method entitles the following: a) synthesis of a combinatorial library on the HMBA-ChemMatrix resin by the divide-couple-recombine (DCR) method using the Fmoc chemistry, b) library screening with the protein of interest labeled with a fluorescent dye or biotin, c) identification of peptides contained on positive beads by tandem matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS/MS), d) solid phase peptide ligands synthesis and immobilization in chromatographic supports, e) evaluation of protein adsorption on peptide affinity matrices from the equilibrium isotherms and breakthrough curves.