Design of protease-resistant peptide ligands for the purification of antibodies from human plasma.

Journal Article (Journal Article)

A strategy is presented for developing variants of peptide ligands with enhanced biochemical stability for the purification of antibodies from animal sera. Antibody-binding sequences HWRGWV, HYFKFD, and HFRRHL, previously discovered by our group, were modified with non-natural amino acids to gain resistance to proteolysis, while maintaining target affinity and selectivity. As trypsin and α-chymotrypsin were chosen as models of natural proteolytic enzymes, the basic (arginine and lysine) and aromatic (tryptophan, phenylalanine, and tyrosine) amino acids were replaced with non-natural analogs. Using the docking software HADDOCK, a virtual library of peptide variants was designed and screened in-silico against the known HWRGWV binding site on the pFc fragment of IgG. A pool of selected sequences with the highest predicted free energy of binding was synthesized on chromatographic resin, and the resulting adsorbents were tested for IgG binding and resistance to proteases. The ligand variants exhibited binding capacities and specificities comparable to the original sequences, yet with much higher proteolytic resistances. The sequences HWMetCitGWMetV and HFMetCitCitHL was used for purifying polyclonal IgG from IgG-rich fractions of human plasma, with yields and purity above 90%. Notably, due to electrical neutrality, the variant showed higher selectivity than the original sequence. Binding isotherms were also constructed, which confirmed the docking predictions. This method represents a general strategy for enhancing the biochemical stability as well as the affinity and selectivity of natural or synthetic peptide ligands for bioseparations.

Full Text

Duke Authors

Cited Authors

  • Menegatti, S; Bobay, BG; Ward, KL; Islam, T; Kish, WS; Naik, AD; Carbonell, RG

Published Date

  • May 2016

Published In

Volume / Issue

  • 1445 /

Start / End Page

  • 93 - 104

PubMed ID

  • 27072524

Electronic International Standard Serial Number (EISSN)

  • 1873-3778

International Standard Serial Number (ISSN)

  • 0021-9673

Digital Object Identifier (DOI)

  • 10.1016/j.chroma.2016.03.087


  • eng